CN110003344B

CN110003344B - Amino acid optical probe and preparation method and application thereof

Info

Publication number: CN110003344B
Application number: CN201910149304.7A
Authority: CN
Inventors: 杨弋; 赵玉政; 李写; 徐磊; 黄立
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2023-05-05
Anticipated expiration: 2039-02-28
Also published as: CN110003344A

Abstract

The invention relates to an amino acid optical probe, a preparation method and application thereof. In one aspect, the invention relates to an optical probe comprising an amino acid-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or the functional variant thereof is located within the sequence of the amino acid-sensitive polypeptide or the functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in detecting amino acid. The amino acid optical probe provided by the invention has the advantages of relatively small molecular weight, easiness in maturation, large dynamic change of fluorescence, good specificity, capability of expressing in different subcellular organelles of cells, and capability of detecting amino acid in high flux and quantitative inside and outside the cells.

Description

Amino acid optical probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of optical probes, in particular to an amino acid optical probe, a preparation method and application thereof.

Background

The amino acid is a compound in which a hydrogen atom on a carbon atom of a carboxylic acid is substituted with an amino group, and is an organic compound containing a basic amino group and an acidic carboxyl group. The amino acids obtained after protein hydrolysis are all alpha-amino acids with amino groups attached to the alpha-carbon, and only twenty or more are the basic units constituting the protein. Amino acids act metabolically in the human body, for example, by synthesis of tissue proteins; amino-containing substances such as synthetic acids, hormones, antibodies, creatine, etc.; converted into carbohydrates and fats; or oxidized to carbon dioxide and water and urea to produce energy.

Alanine is one of the 20 natural amino acids, which is the basic amino acid constituting the protein. Alanine acts as an optional amino acid and acts in the cell primarily as an N-source donor for glutamate and by a reversible reaction with pyruvate through transamination. Pyruvic acid and glutamic acid are produced between alanine and alpha-ketoglutarate by the action of alanine Aminotransferase (ALT). Alanine metabolism is an indispensable loop in the pyruvate synthesis pathway, so alanine is also considered to be one of the most important amino acids in the gluconeogenesis pathway. ALT in cells can be subjected to an inter-ammonia transfer between pyruvic acid and alanine as required (Jojima T et al, appl Microbiol Biotechnol.2010,87 (1): 159-165; garcia R F et al, amino acids.2007,33 (1): 151-155;Pukrittayakamee S et al, trop Med Int health.2002,7 (11): 911-918; brosnan J T et al, J Biol chem.2001,276 (34): 31876-31882;Arguello J M et al, arch Biochem Biophys.1999,367 (2): 341-347). The ALT enzyme activity reference values in serum of an adult are: 8-50U/L. When lesions occur in various tissues and organs in the human body, ALT is released into the blood along with death of the lesion cells, so that the ALT content in serum is increased. ALT content in serum increases more than one time when one thousandth of hepatocytes produce inflammation, so the serum transaminase amount is an important index of the degree of liver lesions (Oka R et al, J Atheroscler Thromb.2014,21 (6): 582-592; liu Z et al, int J Med Sci.2014,11 (9): 925-935; kunutsor S K et al, PLoS one.2014,9 (4): e 96068). In addition, L-alanine is an important raw material for synthesizing vitamin B6, and the amino acid injection taking L-alanine as a main component has a certain treatment effect on liver and brain diseases, and meanwhile, L-alanine is also a good diuretic. Recent studies have shown that alanine co-glucose can increase kanamycin killing the multidrug-resistant strain Edwardsiella tarda, and that alanine co-glucose increases NADH production through tricarboxylic acid cycle and increases transmembrane proton dynamics in bacteria, thereby increasing kanamycin uptake and killing of bacteria (Peng B et al, cell Metab.2015,21 (2): 249-261).

The detection of the alanine content is also particularly important, since alanine has the above-mentioned important role. Common detection methods for alanine are capillary electrophoresis (Li X-t et al, chem Res Chin Univ 2013,29 (3): 434-438; meng J et al, the analysis 2010,135 (7): 1592-1599), high performance liquid chromatography (Tateda N et al, analytical sciences: the international journal of The Japan Society for Analytical Chemistry 2001,17 (6): 775-778), ultraviolet-visible spectrophotometry (Du J et al, chemical communications (Cambridge, england) 2013,49 (47): 5399-5401; engeser M et al, chemical Communications, (13): 1191-1192) and fluorescence spectrometry (Engeser M et al, chemical Communications 1999, (13): 1191-1192).

Proline is widely distributed in various animals and plants. Proline accumulation is a very common and important phenomenon in plants. When the plant is endangered by the external environment, the proline content in the plant body can be increased drastically, and the mechanism of this phenomenon and the physiological effect thereof in the plant body are still not studied. In mammalian cells, however, the physiological role of proline as an optional amino acid has not been widely recognized at the beginning, but rather it is a raw material for protein synthesis and has an important role in maintaining the framework structure of proteins due to its special structure. However, in recent years, along with the discovery of the regulation of intracellular ROS by the proline metabolic pathway, attention has been focused on the study of proline and its metabolic pathway in mammalian cells.

Proline metabolism in mammals occurs primarily in the cytosol and mitochondria, primarily proline enters the mitochondria by a transport protein on the mitochondria, oxidative dehydrogenation of proline by a proline oxidase (POX/PRODH) on the inner mitochondrial membrane to Δ1-pyrroline-5-carboxylic acid (P5C), and P5C shuttles back to the cytosol and converts back to proline by PYCR (P5C reductase) in the cytosol. This results in a cycle of proline metabolism. Δ1-pyrroline-5-carboxylic acid (P5C) can form a tautomeric equilibrium with GSA (glutamate- γ -semialdehyde) in the cytoplasm, finally GSA can be metabolized by P5CDH dehydrogenation to glutamate into the tricarboxylic acid cycle on the one hand and ornithine transaminase to ornithine into the urea cycle on the other hand. In general terms, the metabolic pathways formed by the regulation of P5C and proline interconversions between cytoplasm and mitochondria by POX and PYCR (P5 CDH) are referred to as the proline cycle (Liu W et al, biofactor.2012, 38 (6): 398-406; liu W et al autophagy.2012,8 (9): 1407-1409; liu W et al, cancer res.2012,72 (14): 3677-3686).

In recent years, it has been found that the Proline Oxidase (POX) gene can negatively regulate the growth of tumor cells by promoting cell cycle arrest, inducing cell differentiation, promoting apoptosis and the like, and that abnormal expression thereof is found in various tumors. The oxidative dehydrogenation of proline by POX also consumes ADP to produce ATP and a free electron which is accompanied by the production of ROS (reactive oxygen species) which are critical controls of apoptosis, proliferation and cell cycle in the process of transmission in the electronic respiratory chain in the centromere (Liu Y et al, cancer Res.2009,69 (16): 6414-6422; phng J M et al, szabados L et al, trends Plant Sci.2010,15 (2): 89-97; pandhare J et al, J Biol chem.2006,281 (4): 2044-2052; liu Y et al, oncogene.2006,25 (41): 5640-5647).

Common detection methods for proline include acidic ninhydrin chromogenic method (Chen et al, applie Environmental Microbiology 2006, 72:4001-4006), spectrophotometry (Hortala MA et al, J Am Chem So2003,125 (1): 20-21; puF et al, anal Chem 2010,82 (19): 8211-8216), high performance liquid chromatography (Wadud S et al, journal of chromatography B, analytical technologies in the biomedical and life sciences 2002,767 (2): 369-374), and the like.

Valine, leucine and isoleucine are all branched-chain amino acids and are all essential amino acids for human body. The three work together to promote normal growth of the body, repair tissues, regulate blood sugar and provide the required energy. Valine can provide additional energy to muscles to produce glucose when engaged in intense physical activity to prevent muscle weakness. It also helps to remove excess nitrogen from the liver and transport the body's required nitrogen to various sites. Leucine increases the production of growth hormone and helps burn visceral fat which, because of its internal body, is difficult to work effectively with just diet and exercise. Injections of branched-chain amino acids such as valine are commonly used to treat liver failure and damage to these organs caused by alcoholism and drug abuse. In addition, it can be used as a therapeutic agent for accelerating wound healing. Common detection methods for valine, leucine and isoleucine are acid-base titration and potentiometric titration.

Serine is a non-essential amino acid that plays a role in the metabolism of fat and fatty acids and in the growth of muscles. Serine it contributes to the production of immune hemagglutinin and antibodies, serine is also required to maintain a healthy immune system. Serine plays a role in the processing of cell membranes, muscle tissue and the synthesis of sheaths around nerve cells. Common detection methods for serine include spectrophotometry, fluorescence quenching, ninhydrin color development, high performance liquid chromatography, enzyme reaction detection, and capillary electrophoresis-electrochemiluminescence (CE-ECL) method (Li Zhongcai, et al, research progress in L-serine detection technology, industrial microorganism, 2016 (5): 61-65).

Threonine is also an essential amino acid and is mainly used in medicine, chemical reagent, food enhancer, feed additive and the like. Threonine is an amino acid which is converted into other substances by the catalysis of Threonine Dehydratase (TDH) and Threonine Dehydratase (TDG) and aldolase, unlike other amino acids, which are not dehydrogenases and transaminases. The route is mainly 3: metabolizing glycine and acetaldehyde by aldolase; metabolizing into aminopropionic acid, glycine and acetyl COA by TDG; is metabolized by TDH into propionic acid and alpha-aminobutyric acid. Threonine is an important nutrition enhancer, and has the effects of relieving fatigue of human body and promoting growth and development like tryptophan. In medicine, because threonine contains hydroxyl in the structure, the threonine has water-holding effect on human skin, is combined with oligosaccharide chains, plays an important role in protecting cell membranes, and can promote phospholipid synthesis and fatty acid oxidation in vivo. Common detection methods for threonine include amino acid analysis, paper chromatography, ninhydrin method, and formaldehyde method.

Cysteine is the only amino acid having a reducing group thiol (-SH) among 20 or more amino acids constituting a protein, and it is involved in the cell reduction process and the synthesis of the protein, glutathione in the organism (Yang P. Et al, sensitive chemiluminescencemethod for the determination of glutathione, L-cysteine and 6_mercaptoprorine [ J ]. Microchem Acta,2008 (163): 263-269). SH can form insoluble thiolate with Ag+, hg+, cu2+ and other metal ions, and can be condensed with toxic aromatic compounds into thioether amino acid to play a role in detoxification, and the SH can be mutually converted with disulfide bonds contained in cystine, so that the cysteine can be easily oxidized to form disulfide bonds, the two cysteine states forming disulfide bonds are usually called oxidation states, and the cysteine not forming disulfide bonds is called reduction states, so that L-cysteine has strong reducibility. Since L-cysteine has an isoelectric point close to neutral pH, a thiol group is generally present and has a high activity, and thus has many biological functions such as enhancing liver function, resolving phlegm, promoting hair growth, preventing oxidation of food, etc., and is now widely used in medicine, food, cosmetics, and feed industries. Thus, studies on L-cysteine, including quantitative analysis, have attracted widespread interest and are of particular importance.

The method for measuring cysteine includes a colorimetric method, a titration method, a chromatography method (Ma Guzhou, etc.) of classical methods, an analytical method [ M ] for additives in foods, food chemical lessons of environmental sanitation office, tokyo, japan, beijing: chinese Standard Press publication, 1988, 436), optical rotation (Zhang Weiguo, li. Optical rotation determination of L-cysteine content [ J ]. North China coal medical college, 2001,3 (4): 435), fluorescence (Cao Qiue, li Fei. Flow injection fluorometry method for cysteine in medicine, J ]. Yunnan university, 2003, 25 (3): 266-268), single scanning polarography (Zhao Cheng, cui Jimao, chen Song. Single scanning polarography for determination of cystine and cysteine [ J ]. Professional health and disease, 2004, 19 (3): 206), electrochemical (Hsiao Y., su ff., cheng J., et al, electrochemical determinationof cysteine based on conducting polymers gold nanopar-ticles hybridnanocomposites [ J ]. Electrochimica Acta,2011, 56 (20): 6887-6895), high performance liquid chromatography (Zheu. H., maG., et al, detection of S-32-24-d, and 6-handicap.2011-3975, and 2011-37-58-J, 6-25-6-J, and 2011-37-6-J, 6-handicap). The above methods either require complicated sample extraction and refinement or require high equipment requirements and are expensive.

In summary, these amino acid detection methods of the prior art are not suitable for living cell studies, and suffer from a number of drawbacks: time-consuming sample processing procedures such as cell disruption, separation, extraction and purification, etc. are required; in situ, real-time, dynamic, high throughput and high spatial-temporal resolution detection in living cells and subcellular organelles is not possible. There remains a need in the art for methods that allow for the real-time localization, quantification, high throughput detection of alanine both inside and outside the cell.

Disclosure of Invention

The invention aims to provide a probe and a method for the real-time localization, high-throughput and quantitative detection of amino acids inside and outside cells.

In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, the present invention provides an amino acid optical probe comprising an amino acid sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or functional variant thereof is located within the sequence of the amino acid sensitive polypeptide or functional variant thereof. The amino acid-sensitive polypeptide or functional variant thereof is separated into a first portion and a second portion by the optically active polypeptide or functional variant thereof.

The invention provides an amino acid optical probe, which comprises an amino acid sensitive polypeptide B and an optical active polypeptide A, wherein the optical active polypeptide A is positioned in the sequence of the amino acid sensitive polypeptide B, and the amino acid sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of a B1-A-B2 type.

In one embodiment, the amino acid-sensitive polypeptide includes an amino acid binding protein and functional variants thereof. In one embodiment, the amino acid binding protein is derived from agrobacterium, e.g., agrobacterium fabrum. In one embodiment, the amino acid binding protein is derived from Atu2422-GABA receptor protein or an analog thereof. In one embodiment, the amino acid binding protein comprises a Atu2422-GABA receptor protein or functional variant thereof. In one embodiment, the amino acid binding protein comprises an alanine binding protein. In a specific embodiment, the amino acid binding protein has the sequence shown in SEQ ID NO. 1 or a functional variant thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity thereto.

In one embodiment, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one embodiment, the fluorescent protein is selected from the group consisting of yellow fluorescent protein (cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (mKate as shown in SEQ ID NO:4 or 8, mcherry as shown in SEQ ID NO: 5), green fluorescent protein (cpGGFP as shown in SEQ ID NO: 6), blue fluorescent protein (cpbFP as shown in SEQ ID NO: 7), apple red fluorescent protein (cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one embodiment, the fluorescent protein has the sequence shown in any one of SEQ ID NOs 2-9.

In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the invention may be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by no more than 5 amino acid linkers, e.g., 0, 1, 2, 3, 4 amino acid linkers. In one embodiment, the linker is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker. In one embodiment, the optical probe is as follows: first portion B1 of the amino acid-sensitive polypeptide-optically active polypeptide a-second portion B2 of the amino acid-sensitive polypeptide.

In one embodiment, the optical probes of the invention further comprise a localization sequence for localizing the probe to a specific organelle, e.g., a cell.

The optically active polypeptides of the invention may be located at or fused to any position of the amino acid sensitive polypeptides described herein. In one embodiment, the optically active polypeptide is located within a segment of an amino acid-sensitive polypeptide described herein selected from the group consisting of: residues 117-123, residues 249-259, and residues 323-330, numbering corresponds to the full length of the amino acid susceptible polypeptide. In one embodiment, the optically active polypeptide replaces one or more amino acids within a segment of the amino acid-sensitive polypeptide described herein selected from the group consisting of: residues 117-123, residues 249-259, and residues 323-330, numbering corresponds to the full length of the amino acid susceptible polypeptide.

In one embodiment, the optically active polypeptide is located at a site of the amino acid-sensitive polypeptide described herein selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256, 254/257, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 and 329/330. Herein, if two numbers in a site represented in the form of "X/Y" are consecutive integers, it means that an optically active polypeptide is located between the amino acids represented by the numbers. For example, the position at position 117/118 indicates that the optically active polypeptide is located between amino acids 117 and 118 of the amino acid sensitive polypeptides described herein. If two numbers in the site represented in the form of "X/Y" are not consecutive integers, it is meant that the optically active polypeptide replaces the amino acid between the amino acids indicated by the numbers. For example, positions 249/259 indicate that the optically active polypeptide replaces amino acids 250-258 of the amino acid-sensitive polypeptides described herein. Preferably, the optically active polypeptide is located at the following positions of the amino acid-sensitive polypeptide described herein: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330.

In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be a probe formed when cpYFP is located at 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 of Atu2422 as shown in SEQ ID NOS 10-15. In one embodiment, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NOS 10-15. In one embodiment, the optical probe is sensitive to an amino acid selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

The invention also provides amino acid-sensitive polypeptides described herein having one or more mutations, including amino acid-binding proteins having one or more mutations. The mutation comprises modification, substitution, deletion of amino acids or truncation of sequences. The mutation may be selected from the group consisting of mutations at the F77, A100, T102, D121, Y150, D226, G227, and Y275 isosteres of the amino acid sensitive polypeptide. Illustratively, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the mutation is selected from the group consisting of F77A, F77L, a100G, D121E, D121T, D121V, D226E, D226N, G227S, and Y275F.

Amino acid-sensitive polypeptides (e.g., amino acid binding proteins) in the optical probes of the invention may comprise one or more amino acid mutations. In some embodiments, the amino acid-sensitive polypeptide in the optical probe comprises an amino acid binding protein having one or more mutations described herein. In one embodiment, the mutation is, for example, a mutation at a site selected from the group consisting of F77, A100, T102, D121, Y150, D226, G227, and Y275 of the amino acid sensitive polypeptide. In one embodiment, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the amino acid-sensitive polypeptide in the optical probe of the invention may comprise a mutation selected from the group consisting of: F77A, F77L, a100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

In exemplary embodiments, the B1-A-B2 type optical probe of the present invention may be a probe of Atu2422 fused with cpYFP at position 121/122 and having mutations of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S, or Y275F as shown in SEQ ID NOS.16-25. In one exemplary embodiment, the optical probes of the present invention have or consist of the sequences shown in SEQ ID NOS.16-25. In one embodiment, the optical probe comprising one or more amino acid mutations is sensitive to alanine.

The optical probe provided by the invention comprises any one of amino acid sequences SEQ ID NO 10-25 or variants thereof. In one embodiment, the invention provides an optical probe comprising a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOs 10-25. In a preferred embodiment, the optical probes provided herein comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10-25.

The invention also provides a detection kit comprising an amino acid optical probe or fusion polypeptide as described herein or an amino acid optical probe or fusion polypeptide prepared as described herein. In one embodiment, the kit detects an amino acid selected from the group consisting of: alanine, proline, valine, serine, isoleucine, threonine and cysteine.

The present invention provides a method of making an optical probe as described herein comprising: providing a cell comprising a vector expressing an optical probe or fusion polypeptide described herein, culturing the cell under conditions in which the cell expresses, and isolating the optical probe or fusion polypeptide. In one embodiment, a method of making an amino acid optical probe or fusion polypeptide described herein comprises: 1) Transferring an expression vector encoding an amino acid optical probe described herein into a host cell; 2) Culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the amino acid optical probe.

The invention also provides a method of detecting an amino acid in a sample comprising: contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular or in situ. Such as blood. In one embodiment, the amino acid in the sample is selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

Also provided herein are methods of quantifying amino acids in a sample, comprising: contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, detecting a change in an optically active polypeptide, and quantifying an amino acid in the sample based on the change in the optically active polypeptide. In one embodiment, the amino acid in the sample is selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine.

The invention also provides a method of screening a compound (e.g., a drug) comprising: contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a candidate compound, detecting a change in an optically active polypeptide, and screening the compound for a change in the optically active polypeptide. The method can screen compounds with high throughput.

The invention also provides the use of an amino acid optical probe or fusion polypeptide as described herein or prepared by a method as described herein in the real-time localization of an amino acid. In one embodiment, the real-time localization of the amino acid comprises real-time localization of an amino acid selected from the group consisting of: alanine, proline, valine, serine, isoleucine, threonine and cysteine.

In a second aspect, the invention provides an alanine-optical probe comprising an alanine-sensitive polypeptide, or a functional variant thereof, and an optically-active polypeptide, or a functional variant thereof, wherein the optically-active polypeptide, or functional variant thereof, is located within the sequence of the alanine-sensitive polypeptide, or functional variant thereof. The alanine-sensitive polypeptide or functional variant thereof is separated into a first portion and a second portion by the optically active polypeptide or functional variant thereof.

The invention provides an alanine optical probe, which comprises an alanine-sensitive polypeptide B and an optical active polypeptide A, wherein the optical active polypeptide A is positioned in the sequence of the alanine-sensitive polypeptide B, and the alanine-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of a B1-A-B2 formula.

In one embodiment, the alanine-sensitive polypeptide includes an amino acid binding protein or a functional variant thereof. In one embodiment, the amino acid binding protein is derived from agrobacterium, e.g., agrobacterium fabrum. In one embodiment, the amino acid binding protein is derived from Atu2422-GABA receptor protein or an analog thereof. In one embodiment, the amino acid binding protein comprises a Atu2422-GABA receptor protein or functional variant thereof. In one embodiment, the amino acid binding protein comprises an alanine binding protein. In a specific embodiment, the amino acid binding protein has the sequence shown in SEQ ID NO. 1 or a functional variant thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity thereto.

In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the invention may be any amino acid sequence of any length. In one embodiment, the optically active polypeptide is flanked by no more than 5 amino acid linkers, e.g., 0, 1, 2, 3, 4 amino acid linkers. In one embodiment, the linker is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker. In one embodiment, the optical probe is as follows: first portion of alanine-sensitive polypeptide B1-optically active polypeptide A-second portion of alanine-sensitive polypeptide B2.

The optically active polypeptides of the invention can be located at or fused to any position of the alanine-sensitive polypeptide. In one embodiment, the optically active polypeptide is located within a segment of an alanine-sensitive polypeptide selected from the group consisting of: residues 117-123, residues 249-259, and residues 323-330, numbering corresponds to the full length of the alanine-sensitive polypeptide. In one embodiment, the optically active polypeptide replaces one or more amino acids within a segment of an alanine-sensitive polypeptide selected from the group consisting of: residues 117-123, residues 249-259, and residues 323-330, numbering corresponds to the full length of the alanine-sensitive polypeptide.

In one embodiment, the optically active polypeptide is located at a site selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256, 254/257, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 and 329/330. Herein, if two numbers in a site represented in the form of "X/Y" are consecutive integers, it means that an optically active polypeptide is located between the amino acids described by the numbers. For example, the position at position 117/118 indicates that the optically active polypeptide is located between amino acids 117 and 118 of the alanine-sensitive polypeptide. If two numbers in the site represented in the form of "X/Y" are not consecutive integers, it is meant that the optically active polypeptide replaces the amino acid between the amino acids indicated by the numbers. For example, positions 249/259 indicate that the optically active polypeptide replaces amino acids 250-258 of the alanine-sensitive polypeptide. Preferably, the optically active polypeptide is located at the following positions of the alanine-sensitive polypeptide: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330.

In an exemplary embodiment, the B1-A-B2 type optical probe of the present invention may be a probe formed when cpYFP is located at 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 of Atu2422 as shown in SEQ ID NOS 10-15. In one embodiment, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NOS 10-15.

The invention also provides alanine-sensitive polypeptides having one or more mutations, including amino acid binding proteins having one or more mutations. The mutation comprises modification, substitution, deletion of amino acids or truncation of sequences. The mutation may be selected from the group consisting of mutations at the F77, A100, T102, D121, Y150, D226, G227, and Y275 isosteres of the alanine-sensitive polypeptide. Illustratively, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the mutation is selected from the group consisting of F77A, F77L, a100G, D121E, D121T, D121V, D226E, D226N, G227S, and Y275F.

The alanine-sensitive polypeptide (e.g., amino acid binding protein) in the optical probes of the invention can comprise one or more mutations. In some embodiments, the alanine-sensitive polypeptide in the optical probe includes an amino acid binding protein having one or more mutations described herein. In one embodiment, the mutation is, for example, a mutation at a site selected from the group consisting of F77, A100, T102, D121, Y150, D226, G227, and Y275 of an alanine-sensitive polypeptide. In one embodiment, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the alanine-sensitive polypeptide in the optical probe of the present invention can include a mutation selected from the group consisting of: F77A, F77L, a100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F.

In exemplary embodiments, the B1-A-B2 type optical probe of the present invention may be a probe of Atu2422 fused with cpYFP at position 121/122 and having mutations of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S, or Y275F as shown in SEQ ID NOS.16-25. In one embodiment, the optical probes of the invention have or consist of the sequences shown in SEQ ID NOS.16-25.

The optical probe provided by the invention comprises any one of amino acid sequences SEQ ID NO 10-25 or variants thereof. In one embodiment, the invention provides an optical probe comprising a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOs 10-25. In a preferred embodiment, the optical probes provided herein comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10-25. In a more preferred embodiment, the optical probe provided by the invention comprises or consists of SEQ ID NO. 24.

The invention also provides fusion polypeptides comprising an optical probe as described herein and other polypeptides. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptide is located at the N-terminus and/or the C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, tags for purification, or tags for immunoblotting. The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.

Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, golgi apparatus, lysosomes, peroxisomes, and the like. In some embodiments, the tag for purification or for immunoblotting comprises 6 histidine (6 xhis), glutathione-s-transferase (GST), flag.

The invention also provides nucleic acid sequences encoding the optical probes or fusion polypeptides described herein, or the complements thereof. In one embodiment, the invention provides a nucleic acid sequence encoding an amino acid sequence as set forth in any one of SEQ ID NOs 10-25. In one embodiment, the nucleic acid sequences of the invention comprise any one of the nucleotide sequences SEQ ID NOS.26-27 or variants thereof. In a preferred embodiment, the invention provides a nucleic acid sequence comprising a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any one of the nucleotide sequences SEQ ID NOS.26-27. In another preferred embodiment, the invention provides a nucleic acid sequence comprising a nucleotide sequence substantially similar or identical to any one of nucleotide sequences SEQ ID NOs 26 to 27.

The invention also relates to the complementary sequences of the above-mentioned nucleic acid sequences or variants thereof, which may comprise nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the optical probes or fusion proteins of the invention or the complementary sequences thereof.

The amino acid sequences and nucleic acid sequences in the present invention are preferably provided in isolated form, more preferably purified to homogeneity.

The invention also provides an expression vector comprising a nucleic acid sequence of the invention or a complement thereof, encoding an optical probe or fusion polypeptide of the invention, operably linked to an expression control sequence. In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector. In some embodiments, the prokaryotic expression vector is preferably obtained from the operative ligation of plasmid pRSETb with the nucleic acid sequences described herein. In some embodiments, the expression control sequences include an origin of replication, a promoter, an enhancer, an operon, a terminator, and a ribosome binding site.

The invention also provides a cell comprising an expression vector of the invention comprising a nucleic acid sequence of the invention or a complement thereof operably linked to an expression control sequence. The cells express the optical probes or fusion polypeptides described herein.

The invention also provides a detection kit comprising an alanine optical probe or fusion polypeptide as described herein or prepared as described herein.

The present invention provides a method of making an optical probe as described herein comprising: providing a cell comprising a vector expressing an optical probe or fusion polypeptide described herein, culturing the cell under conditions in which the cell expresses, and isolating the optical probe or fusion polypeptide. In one embodiment, a method of making an alanine optical probe or fusion polypeptide described herein comprises: 1) Transferring an expression vector encoding an alanine optical probe as described herein into a host cell; 2) Culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the alanine optical probe.

The invention also provides a method of detecting alanine in a sample comprising: contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular or in situ. Such as blood.

Also provided herein are methods of quantifying alanine in a sample, comprising: contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, detecting a change in an optically active polypeptide, and quantifying alanine in the sample based on the change in the optically active polypeptide.

The invention also provides the use of an alanine optical probe or fusion polypeptide as described herein or prepared as described herein in the real-time localization of alanine.

The invention has the beneficial effects that: the amino acid optical probe provided by the invention is easy to mature, large in fluorescence dynamic change and good in specificity, can be expressed in cells by a gene operation method, can be used for positioning, high-flux and quantitative detection of amino acids inside and outside the cells in real time, and omits the time-consuming step of processing samples. The experimental effect shows that the highest response of the amino acid optical probe provided by the application to the amino acid reaches more than 4 times of that of a control, and the cell can be positioned, qualitatively and quantitatively detected in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, golgi apparatus and the like, and the high-throughput compound screening and the quantitative detection of the amino acid in blood can be performed.

Drawings

The invention is further described below with reference to the drawings and examples.

FIG. 1 is a SDS-PAGE diagram of an exemplary amino acid optical probe as described in example 1;

FIG. 2 is a graph of the response of an exemplary amino acid optical probe comprising cpYFP and an amino acid binding protein to alanine as described in example 2;

FIG. 3 is a graph of changes in alanine response of an exemplary amino acid optical probe comprising a CPGFP and an amino acid binding protein described in example 3;

FIG. 4 is a graph of the response of an exemplary amino acid optical probe comprising a cpBFP and an amino acid binding protein to alanine as described in example 4;

FIG. 5 is a graph of the change in alanine response of an exemplary amino acid optical probe comprising cpm apple and an amino acid binding protein described in example 5;

FIG. 6 is a graph showing titration curves for amino acid optical probes fused to cpYFP at positions 120/121, 121/122, 121/123, 324/330, 325/330 or 326/330 of an amino acid binding protein for different concentrations of alanine as described in example 6;

FIG. 7A is a bar graph of an exemplary amino acid optical probe fused to cpYFP at positions 121/122 of an amino acid binding protein for specific detection of 20 amino acids as described in example 6;

FIG. 7B is a bar graph of the specificity of the probe for alanine at position 121/122 of the amino acid binding protein for a fluorescent protein as compared to the fusion protein fused to either the N-or C-terminus of the amino acid binding protein as described in example 6;

FIG. 8 is a bar graph of an exemplary amino acid photo probe to alanine response at amino acid binding protein positions 121/122 fused to cpYFP and having mutations at positions F77, A100, T102, D121, Y150, D226, G227 or Y275 as described in example 7;

FIG. 9 is a graph of fluorescence spectrum properties of an exemplary amino acid optical probe described in example 8;

FIG. 10 is a graph showing titration curves for different concentrations of alanine for an exemplary amino acid optical probe described in example 8;

FIG. 11 is a bar graph of the specific detection of 20 amino acids by an exemplary amino acid optical probe described in example 8;

FIG. 12 is a photograph of subcellular organelle localization of an exemplary amino acid optical probe in mammalian cells according to example 9;

FIG. 13 is a schematic representation of dynamic monitoring of alanine transmembrane transport of an exemplary amino acid optical probe in different subcellular organelles in a mammalian cell as described in example 9;

FIG. 14 is a plot of high throughput compound screening at the living cell level for an exemplary amino acid optical probe described in example 10;

FIG. 15 is a bar graph of the quantification of alanine in mouse and human blood by an exemplary amino acid optical probe described in example 11.

Detailed Description

When a value or range is given, the term "about" as used herein means that the value or range is within 20%, within 10% and within 5% of the given value or range.

The terms "comprising," "including," and equivalents thereof as used herein include the meaning of "containing" and "consisting of … …," e.g., a composition "comprising" X may consist of X alone or may contain other substances, e.g., x+y.

The term "amino acid-sensitive polypeptide" or "amino acid-responsive polypeptide" as used herein refers to a polypeptide that responds to an amino acid, including any response in a chemical, biological, electrical or physiological parameter of the polypeptide that is associated with the interaction of the sensitive polypeptide. The amino acid-sensitive polypeptide is sensitive to any amino acid that forms a protein. In one embodiment, the amino acid-sensitive polypeptide is sensitive to an amino acid selected from the group consisting of alanine, proline, valine, serine, isoleucine, threonine, and cysteine. Responses include small changes, e.g., changes in the orientation of amino acids or peptide fragments of a polypeptide, e.g., changes in the primary, secondary, or tertiary structure of a polypeptide, including, e.g., changes in protonation, electrochemical potential, and/or conformation. A "conformation" is a three-dimensional arrangement of primary, secondary and tertiary structures of a molecule comprising pendant groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include a transition from an alpha-helix to a beta-sheet or from a beta-sheet to an alpha-helix. It will be appreciated that the detectable change need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered. Amino acid-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of an amino acid-sensitive polypeptide include, but are not limited to, variants that can interact with an amino acid to effect the same or similar change as the parent amino acid-sensitive polypeptide. An "amino acid susceptible polypeptide" can be any amino acid susceptible polypeptide, such as an alanine-susceptible polypeptide, including amino acid binding proteins. Illustratively, the term "alanine-sensitive polypeptide" or "alanine-responsive polypeptide" as used herein refers to a polypeptide that responds to amino acids including alanine, a "proline-sensitive polypeptide" or "proline-responsive polypeptide" refers to a polypeptide that responds to amino acids including proline, and so forth.

Amino acid sensitive polypeptides of the invention include, but are not limited to, amino acid binding proteins (amino acid binding protein, AABP) or variants having more than 90% homology thereto. The amino acid binding proteins of the invention may be derived from Agrobacterium, for example Agrobacterium fabrum. An exemplary amino acid binding protein Atu2422 of the invention is an ABC transporter family consisting of two domains linked by three flexible amino acid peptide chains. Amino acid binding proteins can sense changes in amino acid (e.g., alanine) concentration, and the spatial conformation of the amino acid binding protein can change during dynamic changes in amino acid (e.g., alanine) concentration.

The term "optical probe" as used herein refers to an amino acid-sensitive polypeptide fused to an optically active polypeptide. The inventors have found that conformational changes resulting from binding of an amino acid sensitive polypeptide, such as an amino acid binding protein, to a physiological concentration of an amino acid (e.g., alanine) can result in a conformational change of an optically active polypeptide (e.g., a fluorescent protein), which in turn results in a change in the optical properties of the optically active polypeptide. The presence and/or level of an amino acid (e.g., alanine) can be detected and analyzed by plotting a standard curve from the fluorescence of the fluorescent protein measured at different amino acid (e.g., alanine) concentrations. Exemplary Atu2422 proteins are shown in SEQ ID NO. 1. When describing the optical probes of the invention (e.g., when describing the site or mutation site at which the optically active polypeptide is located), reference is made to SEQ ID NO:1 for the amino acid residue number. However, the person skilled in the art knows the corresponding residue numbers of other similar amino acid binding proteins. The amino acid optical probes described herein can be alanine optical probes.

In the optical probes of the invention, an optically active polypeptide (e.g., a fluorescent protein) is operably fused to an amino acid-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used as a means to detect the responsiveness of an optical probe of the invention. As used herein, the term "fluorescent properties" refers to molar extinction coefficient, fluorescence quantum efficiency, shape of excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of excitation at two different wavelengths, emission amplitude ratio at two different wavelengths, excited state lifetime or fluorescence anisotropy at an appropriate excitation wavelength. The measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrates of the invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a particular wavelength or the integration of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are readily distinguishable in the unactivated and activated conformational state. Optically active polypeptides described herein can also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can undergo a change in the same or similar fluorescent properties as the parent optically active polypeptide.

"linker" or "junction region" refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the invention. Illustratively, the amino acid number of the amino terminal of the linker region of the amino acid-sensitive polypeptide and the optically active polypeptide of the present invention is selected to be 0 to 3, and the amino acid number of the carboxy terminal is selected to be 0 to 2; when the recombinant optical probe is linked as a basic unit to a functional protein, it may be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The linker sequence may be a short peptide chain consisting of one or more flexible amino acids.

As used herein, the terms "chromophore", "fluorophore" and "fluorescent protein" are synonymous and refer to proteins that fluoresce upon irradiation with excitation light. The fluorescent protein is used as a basic detection means in the field of bioscience, such as green fluorescent protein GFP and cyclic rearranged blue fluorescent protein (cpBP) derived from mutation of the protein, cyclic rearranged green fluorescent protein (cpGGP), cyclic rearranged yellow fluorescent protein (cpYFP) and the like; also, red fluorescent protein RFP commonly used in the art, and cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate, etc. Exemplary fluorescent proteins have the sequence shown in any one of SEQ ID NOS.2-9.

Green fluorescent protein GFP was originally extracted from Aequorea victoria (Aequorea Victoria), and consisted of 238 amino acids with a molecular weight of about 26kDa. GFP is a unique barrel-like structure formed from 12 beta-sheet chains, in which a chromogenic tripeptide (Ser 65-Tyr66-Gly 67) is entrapped. When in the presence of oxygen, it spontaneously forms the chromophore structure of p-hydroxyphenylmethylene imidazolidinone to fluoresce. GFP fluorescence does not require cofactors and is very stable, a good imaging tool. GFP has two excitation peaks, a main peak at 395nm producing 508nm emission, and excitation light at 475nm at shoulder producing 503nm emission. Exemplary cpGFP is shown as SEQ ID NO:6

Yellow fluorescent protein YFP is derived from green fluorescent protein GFP, and the amino acid sequence of the yellow fluorescent protein YFP has the homology of more than 90 percent with GFP, and the YFP is changed from the key to GFP by mutating the 203 th amino acid from threonine to tyrosine (T203Y). The wavelength of the major excitation peak of YFP was red shifted to 514nm and the emission wavelength was changed to 527nm compared to the original AvGFP. On the basis, the 65 th amino acid of YFP is subjected to site-directed mutagenesis (S65T) to obtain fluorescence-enhanced yellow fluorescent protein EYFP. The cpYFP is obtained by connecting the original N end and C end of GFP through a flexible short peptide chain, manufacturing a new N end and C end at the original GFP near chromophore position, taking the 145 th to 238 th amino acid part as the N end of the new protein, taking the 1 st to 144 th amino acid part as the C end of the new protein, and connecting the two fragments through 5 to 9 flexible short peptide chains. In the present invention, the near chromophore positions are preferably at amino acids Y144 and N145; the short peptide chain having flexibility is preferably VDGGSGGTG or GGSGG. The sequence of an exemplary cpYFP is shown in SEQ ID NO. 2.

Red fluorescent protein RFP was originally extracted from corals in the ocean, wild RFP was an oligomeric protein that was detrimental to fusion expression by organisms, and then red fluorescent proteins of different colorbands were further derived on the basis of RFP, with mCherry and mKate being the most common. Exemplary cpmKates are shown in SEQ ID NO. 4 or 8. An exemplary mCherry is shown in SEQ ID NO. 5.

In other embodiments, the fluorescent protein can be one or more of blue fluorescent protein cpBFP with an amino acid sequence shown in SEQ ID NO. 7, orange fluorescent protein cpmOrange with an amino acid sequence shown in SEQ ID NO. 3, and apple red fluorescent protein cpmApple with an amino acid sequence shown in SEQ ID NO. 9.

The amino acid optical probes of the present invention include amino acid sensitive polypeptides B, such as amino acid binding proteins or variants thereof, and optically active polypeptides a, such as fluorescent proteins. The optical active polypeptide A is inserted into the amino acid sensitive polypeptide B, and the B is divided into two parts of B1 and B2 to form a probe structure of a B1-A-B2 formula; the interaction of amino acid sensitive polypeptide B and amino acid results in a strong optical signal for optically active polypeptide a.

In the optical probes of the invention, the optically active polypeptide may be located at or fused to any position of the amino acid sensitive polypeptide. In one embodiment, the optically active polypeptide is located in the N-C direction at any position of the amino acid-sensitive polypeptide in the N-C direction. In particular, the optically active polypeptide is located in a flexible region of the amino acid sensitive polypeptide, where the flexible region refers to a specific structure, such as a cyclic domain, present in the higher structure of the protein, where the domain has higher mobility and flexibility than other higher structures of the protein, and where the region may undergo a dynamic change in spatial structure conformation upon binding of the protein to the ligand. The flexible region in the present invention mainly refers to the region where the fusion site in the amino acid binding protein is located, such as the regions of amino acid residues 117-123, 249-259 and 317-330. Illustratively, the optically active polypeptide is located at a site of the amino acid sequence of the amino acid binding protein selected from the group consisting of: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256, 254/257, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 and 329/330. In a preferred embodiment, the optically active polypeptide is located at 120/121, 121/122, 121/123, 324/330, 325/330 or 326/330 of the amino acid sequence of the amino acid binding protein. Shown in SEQ ID NO. 10-15.

The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function but different sequences of the polypeptide or protein. These variants include, but are not limited to: sequences obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxy-terminal and/or amino-terminal end thereof. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitution with amino acids having similar or similar properties typically does not alter the function of the polypeptide or protein. Amino acids of similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example. Variants of a polypeptide or protein may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the polypeptide or protein.

The optical probes of the invention may comprise amino acid sensitive polypeptides having mutations. The mutation is for example a mutation at a site selected from the group consisting of F77, A100, T102, D121, Y150, D226, G227 and Y275. Illustratively, the mutation is selected from the group consisting of F77S, F77Y, F77C, F77L, F77P, F77H, F77Q, F77W, F77I, F77T, F77N, F77K, F77R, F77V, F77A, F77D, F77E, F77M, F77A, A100S, A100Y, A100C, A100L, A100P, A100H, A100Q, A100W, A100R, A100I, A100T, A100N, A100K, A100M, A100V, A100F, A100D, A100E, A100G, T102S, T102Y, T102C, T102L, T102P, T102H, T102Q, T102W, T102R, T102I, T102A, T102N, T102K, T102M, T102V, T102F, T102D, T102E, T102G, D121S, D121Y, D121C, D121L, D121P, D121H, D121Q, D121W, D121I, D121T, D121N, D121K, D121R, D121V, D121A, D121F, D121E, D121M, D121A, Y150S, Y150T, Y150C, Y150L, Y150P, Y150H, Y150Q, Y150W, Y150R, Y150I, Y150A, Y150N, Y150K, Y150M, Y150V, Y150F, Y150D, Y150E, Y150G, D226S, D226T, D226C, D226L, D226P, D226H, D226Q, D226W, D226R, D226I, D226A, D226N, D226K, D226M, D226V, D226F, D226Y, D226E, D226G, G227S, G227T, G227C, G227L, G227P, G227H, G227Q, G227W, G227R, G227I, G227A, G227N, G227K, G227M, G227V, G227F, G227Y, G227E, G227D, Y275S, Y275T, Y275C, Y275L, Y275P, Y275H, Y275Q, Y275W, Y275R, Y275I, Y275A, Y275N, Y275K, Y275M, Y275V, Y275F, Y275G, Y275E and Y275D. In one embodiment, the mutation is selected from the group consisting of F77A, F77L, a100G, D121E, D121T, D121V, D226E, D226N, G227S, and Y275F.

In exemplary embodiments, the B1-A-B2 type optical probe of the present invention may be a probe formed when the position 121/122 of Atu2422 is fused with cpYFP and has a mutation selected from the group consisting of F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F, as shown in SEQ ID NOS.16-25.

In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) as compared and aligned for maximum correspondence over a comparison window or designated region, using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25:3389 and Altschul et al (1990) J.mol.biol.215:403, respectively.

The terms "functional variant", "derivative" and "analog" as used herein refer to a protein that retains substantially the same biological function or activity as the original polypeptide or protein (e.g., amino acid binding protein or fluorescent protein). The functional variant, derivative or analogue of a polypeptide or protein of the invention (e.g. an amino acid binding protein or fluorescent protein) may be (i) a protein having one or more, preferably conservative or non-conservative amino acid residues substituted, which may or may not be encoded by the genetic code, or (ii) a protein having a substituent in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g. a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to the protein sequence (e.g. a secretion sequence or a sequence used to purify the protein or a pro-protein sequence, or fusion protein with the formation of an antigen IgG fragment). Such functional variants, derivatives and analogs are within the scope of those skilled in the art, as determined by the teachings herein.

The difference between the analog and the original polypeptide or protein may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, as well as by site-directed mutagenesis or other known molecular biological techniques.

The analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It will be appreciated that the amino acid-sensitive polypeptides of the invention are not limited to the representative proteins, variants, derivatives and analogues listed above. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of proteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

The invention also provides a preparation method of the amino acid optical probe, which comprises the following steps: 1) Incorporating into an expression vector a nucleic acid sequence encoding an amino acid optical probe as described herein; 2) Transferring the expression vector into a host cell; 2) Culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the amino acid optical probe.

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The term "variant" as used herein when referring to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution pattern of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded protein. The nucleic acids of the invention may comprise a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR).

The full-length sequence of the optical probe or fusion protein of the present invention or a fragment thereof can be generally obtained by PCR amplification, artificial synthesis or recombinant methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the nucleotide sequence is more than 2500bp, PCR amplification is preferably performed 2 to 6 times, and then the amplified fragments are spliced together in the correct order. The PCR amplification procedure and system are not particularly limited, and conventional PCR amplification procedures and systems in the art can be adopted. The sequences of interest can also be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating and purifying the relevant polypeptide or protein from the proliferated host cells by conventional methods. Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In the present invention, when the nucleotide sequence of the optical probe is less than 2500bp, the optical probe can be synthesized by adopting an artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in the field, and has no other special requirements. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or functional variants, derivatives or analogues thereof) entirely by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Mutations can be introduced into the protein sequences of the present invention by mutation PCR or chemical synthesis, etc.

After the nucleotide sequence for coding the optical probe is obtained, the nucleotide sequence for coding the optical probe is incorporated into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein to refer to a prokaryotic or eukaryotic vector well known in the art, such as a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors, which are capable of replication and stable expression in a host, an important feature of such recombinant vectors being that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element operably linked to a gene of interest that regulates the transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translational control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc., the choice of expression control sequence being dependent upon the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operably linked" refers to the attachment of a nucleotide sequence of interest to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Methods for constructing expression vectors comprising the fusion protein coding sequences of the invention and appropriate transcriptional/translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses, and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercially available pRSETb vector, with no other special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI, respectively, and then the digested products of both are ligated to obtain a recombinant expression vector. The specific steps and parameters of the digestion and the connection are not particularly limited, and the steps and parameters conventional in the art are adopted.

After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide comprising the fusion protein. Such transfer may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention is a cell capable of receiving and accommodating recombinant DNA molecules, is a site for amplifying recombinant genes, and ideal recipient cells should satisfy both conditions of easy acquisition and proliferation. "host cells" according to the invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples thereof include bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, heLa cells, or Bowes melanoma cells, etc., including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being well known and commonly used in the art. An exemplary host cell for use in the examples of the present invention is E.coli JM109-DE3 strain. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Methods of transferring to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably CaCl ₂ By a method or MgCl ₂ The process is carried out using procedures well known in the art. When the host cell is eukaryotic, the following DNA transfection method may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

After the expression vector is transferred into a host cell, the host cell transferred into the expression vector is amplified, expressed and cultured, and the amino acid optical probe is obtained by separation. The host cell amplification expression culture can be carried out by adopting a conventional method. The medium used in the culture may be various conventional media depending on the kind of host cell used. The culture is carried out under conditions suitable for the growth of the host cell.

In the present invention, the optical probe is expressed in a cell, on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated or purified by various isolation methods using their physical, chemical and other properties. The method for separating the amino acid fluorescent protein is not particularly limited, and a fusion protein separation method conventional in the art can be adopted. Such methods are well known to those skilled in the art and include, but are not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods. In one embodiment, the separation of the optical probe is performed using His-tagged affinity chromatography.

The invention also provides application of the amino acid optical probe in amino acid real-time positioning, quantitative detection and high-flux compound screening. In one aspect, the amino acid optical probe is preferably connected with signal peptides at different parts of the cell, and is transferred into the cell, and the real-time positioning of the amino acid is performed by detecting the intensity of fluorescent signals in the cell; and quantitatively detecting the corresponding amino acid by an amino acid standard dripping curve. The standard amino acid dropping curve is drawn according to fluorescent signals of the amino acid optical probes under the condition of different concentrations of amino acids. The amino acid optical probe is directly transferred into cells, and a time-consuming sample treatment process is not needed in the real-time positioning and quantitative detection process of amino acid, so that the amino acid optical probe is more accurate. When the amino acid optical probe is used for high-flux compound screening, different compounds are added into a cell culture solution, and the change of the amino acid content is measured, so that the compounds with influence on the change of the amino acid content are screened. The application of the amino acid optical probe in the invention in real-time positioning and quantitative detection of amino acid and high-flux compound screening is non-diagnosis and treatment purposes, and does not relate to diagnosis and treatment of diseases. The amino acid may be any amino acid.

Concentrations, amounts, percentages, and other numerical values may be expressed herein in terms of ranges. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, as well as to include all the individual numerical values or sub-ranges encompassed within that range.

Examples

The amino acid optical probes provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

I. Experimental materials and reagents

Examples are mainly conventional methods of cloning in genetically engineered molecular biology, cell culture and imaging, and the like, which are well known to those of ordinary skill in the art, for example: jianluo Skems et al, J. Sambrook, D.W. Lassel, huang Peitang et al: molecular cloning guidelines (third edition, month 8 2002, scientific press publishing, beijing); fei Leixie, et al, basic technical guidelines (fifth edition), zhang Jingbo, xu Cunshuan, et al; j.s. borfepristin, M. darone et al, ind. Cell Biotechnology, zhang Jingbo et al. Modifications and variations as would be apparent to one skilled in the art are within the scope of the claims of this application, as the following examples are not to be construed as being unduly limited to the illustrative embodiments.

pRSETb-cpYFP, pRSETb-amino acid binding protein-based plasmid used in the examples was constructed from the university of Wadong protein laboratory, pRSETb plasmid vector was purchased from Invitrogen corporation. All primers used for PCR were synthesized, purified and identified by mass spectrometry as correct by Shanghai JieRui Bioengineering Co. The expression plasmids constructed in the examples were subjected to sequencing, which was performed by Huada gene company and Jie Li Cexu company. Taq DNA polymerase used in each example was purchased from Dongsheng, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Co., ltd, primestaR DNA polymerase was purchased from TaKaRa Co., ltd, and the three polymerases were all supplemented with the corresponding polymerase buffer and dNTP. BamHI, bglII, hindIII, ndeI, xhoI, ecoRI, speI, T4 ligase, T4 phosphorylase (T4 PNK) are purchased from Fermentas, inc., and corresponding buffers are added thereto. Transfection reagent Lip2000Kit was purchased from Invitrogen company. Amino acids such as alanine were purchased from Sigma. Unless otherwise specified, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich corporation. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco. The 96-well assay blackboard, 384 Kong Yingguang assay blackboard, were purchased from Grenier company.

The DNA purification kit used in the examples was purchased from BBI (Canada), and the ordinary plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Co. Clone strain Mach1 was purchased from Invitrogen. Both the nickel column affinity chromatography column and the desalting column packing were from GE healthcare.

The main instruments used in the examples include: biotek Synergy 2 multifunctional enzyme-labeled instrument (Bio-Tek Co., ltd.) X-15R high-speed cryocentrifuge (Beckman Co., ltd.), microfuge22R bench-type high-speed cryocentrifuge (Beckman Co., ltd.), PCR amplification instrument (Biometra Co., germany), ultrasonic disruption instrument (Ningbo Xinzhi Co., ltd.), nucleic acid electrophoresis instrument (Shencan Bo Co., ltd.), fluorescence spectrophotometer (Varian Co., ltd.), CO ₂ Constant temperature cell incubator (SANYO), inverted fluorescence microscope (Nikon Corp.).

II molecular biology method and cell experiment method

II.1 Polymerase Chain Reaction (PCR):

1. amplification of the fragment of interest PCR:

the method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system of the PCR amplification is shown in Table 1, and the amplification procedure is shown in Table 2.

TABLE 1 PCR amplification reaction System

TABLE 2 PCR amplification procedure

2. Long fragment (> 2500 bp) amplification PCR:

The long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, a technique for obtaining site-directed mutagenesis in the following examples. Reverse PCR primers were designed at the mutation sites, wherein the 5' end of one primer contained the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.

TABLE 3 Long fragment (> 2500 bp) amplification PCR reaction System

TABLE 4 Long fragment (> 2500 bp) amplification PCR amplification procedure

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TABLE 5 Long fragment (> 2500 bp) amplification PCR amplification procedure

II.2 endonuclease cleavage reaction:

the system for double digestion of plasmid vectors is shown in Table 6, where n represents the amount of sterilized ultra-pure water μL added to make the system total volume.

TABLE 6 plasmid vector double enzyme digestion system

II.3 5' -terminal phosphorylation of DNA fragments

The plasmid or genome extracted from the microorganism contains phosphate groups at the terminal, but the PCR product does not, so that the 5' -terminal base of the PCR product needs to be subjected to phosphate group addition reaction, and only DNA molecules containing phosphate groups at the terminal can undergo ligation reaction. The phosphorylation reaction system is shown in Table 7, wherein T4PNK is a shorthand for T4 polynucleotide kinase, and is used for addition reaction of 5' -terminal phosphate group of DNA molecule.

TABLE 7 phosphorylation reaction System

II.4 ligation of the fragment of interest and the vector

The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present invention

1. Blunt end ligation of blunt end short fragments and linearized vectors

The principle of the method is that a blunt end product obtained by PCR carries out phosphorylation reaction on the 5' end of a DNA fragment under the action of T4PNK, and then is connected with a linearized vector under the action of PEG4000 and T4DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

TABLE 8 blunt end fragment ligation reaction System

2. Ligation of DNA fragments containing cohesive ends and vector fragments containing cohesive ends

DNA fragments cleaved by restriction enzymes will typically produce protruding cohesive ends and thus can be ligated to cohesive end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is shown in Table 9, wherein the mass ratio of PCR product fragments to carrier double cleavage products is approximately between 2:1 and 6:1. .

TABLE 9 viscous terminal ligation reaction System

3. Ligation of the product of 5' -phosphorylated DNA fragments by self-cyclization after introduction of site-directed mutagenesis by inverse PCR

The DNA fragment with phosphorylated 5' end is connected with the 3' end and the 5' end of the linearization vector through self cyclization connection reaction to obtain the recombinant plasmid. The self-cyclized ligation reaction system is shown in Table 10.

TABLE 10 self-cyclizing ligation reaction System

II.5 preparation and transformation of competent cells

Preparation of competent cells:

1. single colonies (e.g., mach 1) were picked and inoculated into 5mL LB medium and shaken overnight at 37 ℃.

2. 0.5-1mL of the overnight cultured bacterial liquid is transferred into 50mL of LB culture medium, and cultured for 3 to 5 hours at 37 ℃ and 220rpm until the OD600 reaches 0.5.

3. The cells were pre-chilled in an ice bath for 2 hours.

Centrifuge at 4000rpm at 4.4℃for 10 min.

5. The supernatant was discarded, and the cells were resuspended in 5mL of pre-chilled buffer, and after homogenization, the resuspension buffer was added to a final volume of 50mL.

6. Ice bath for 45 minutes.

The bacteria were resuspended by centrifugation at 4000rpm at 7.4℃for 10 minutes with 5mL of ice-chilled storage buffer.

8. mu.L of bacterial liquid was placed in each EP tube and frozen at-80℃or with liquid nitrogen.

Resuspension buffer CaCl ₂ (100mM)、MgCl ₂ (70mM)、NaAc(40mM)

Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 XCaCl ₂ (1M)、1mL10×MgCl ₂ (700mM)、1mL 10×NaAc(400mM)、4.6mL ddH ₂ O

Transformation of competent cells:

1. 100. Mu.L of competent cells were thawed on an ice bath.

2. Add the appropriate volume of ligation product, gently blow mix, ice bath for 30 minutes. The ligation product is typically added in a volume of less than 1/10 of the competent cell volume.

3. The bacterial liquid is placed into a 42 ℃ water bath for heat shock for 90 seconds, and is quickly transferred into an ice bath for 5 minutes.

4. mu.L of LB was added and incubated for 1 hour at 200rpm on a thermostatic shaker at 37 ℃.

5. The bacterial liquid was centrifuged at 4000rpm for 3 minutes, 200. Mu.L of supernatant was left to blow the bacterial cells evenly, and the cells were spread evenly on the surface of an agar plate containing an appropriate antibiotic, and the plate was inverted overnight in a thermostatic incubator at 37 ℃.

II.6 expression, purification and fluorescence detection of proteins

1. The expression vector (e.g., pRSETb-based amino acid optical probe expression vector) was transformed into JM109 (DE 3) cells, cultured upside down overnight, cloned into 250ml Erlenmeyer flasks from plates, placed in a 37℃shaker at 220rpm to OD=0.4-0.8, added with 1/1000 (v/v) IPTG (1M), and induced to express at 18℃for 24-36 hours.

2. After the induction expression was completed, the cells were collected by centrifugation at 4000rpm for 30 minutes, and the cell pellet was resuspended in 50mM phosphate buffer and sonicated until the cells were clarified. Centrifugation was performed at 9600rpm at 4℃for 20 minutes.

3. The supernatant was purified by self-contained nickel column affinity chromatography to obtain protein, and the protein after nickel column affinity chromatography was then passed through self-contained desalting column to obtain protein dissolved in 20mM MOPS buffer (pH 7.4) or phosphate buffer PBS.

4. After SDS-PAGE identification of the purified proteins, the probes were diluted with assay buffer (100mM HEPES,100mM NaCl,pH 7.3) or phosphate buffer PBS to give a final concentration of 5-10. Mu.M protein solution. Alanine was prepared as a stock solution at a final concentration of 1M using assay buffer (20 mM MOPS, pH 7.4) or phosphate buffer PBS.

5. Mu.l of 5. Mu.M protein solution was incubated at 37℃for 5 minutes, alanine was added and mixed to a final concentration of 100mM, and the light absorption of the protein at 340nm was measured by a multifunctional fluorescent microplate reader.

6. 100 μl of 1 μM protein solution was incubated at 37deg.C for 5 min, and alanine titration was added to determine the fluorescence intensity of 528nm emission after 485nm fluorescence excitation of the protein. The fluorescence excitation and emission measurement of the sample are completed by a multifunctional fluorescence enzyme-labeling instrument.

7. 100 μl of 1 μM protein solution was incubated at 37deg.C for 5 min, alanine was added, and the absorption spectrum and fluorescence spectrum of the protein were determined. The measurement of the absorption spectrum and fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.

II.7 transfection and fluorescence detection of mammalian cells

1. The pCDNA3.1+ based amino acid optical probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000 (Invitrogen) and placed at 37℃with 5% CO ₂ Is cultured in a cell culture incubator. And (4) carrying out fluorescence detection after the exogenous gene is fully expressed for 24-36 hours.

2. After the induction of expression was completed, the adherent HeLa cells were washed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and enzyme-labeled instrument, respectively.

Example 1: amino acid binding protein plasmids

The AABP (here Atu 2422) gene in the Agrobacterium genes was amplified by PCR, and the PCR products were gel-electrophoresed and recovered and digested with BamHI and EcoRI, while the pRSETb vector was subjected to corresponding double digestion. After ligation with T4DNA ligase, machI was transformed with the product, and the transformed MachI was plated on LB plates (ampicillin 100 ug/mL) and incubated overnight at 37 ℃. The growing MachI transformants were subjected to plasmid extraction and PCR identification. The positive plasmid is sequenced correctly and then the subsequent plasmid construction is carried out.

Example 2: expression and detection of cpYFP optical probes at different fusion sites

In this example, the following site fusion cpYFP was selected based on pRSETb-Atu2422 to give the corresponding pRSETb-Atu2422-cpYFP plasmid: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 120/122, 120/123, 121/122, 121/123, 122/123, 249/250, 249/251, 249/252, 249/253, 249/254, 249/255, 249/256, 249/257, 249/258, 249/259, 250/251, 250/252, 250/253, 250/254, 250/255, 250/256, 250/257, 250/258, 250/259, 251/252, 251/253, 251/254, 251/255, 251/258, 251/259, 252/253, 252/254, 252/255, 252/256, 252/257, 252/258, 252/259, 253/254, 253/255, 253/256, 253/257, 253/258, 253/259, 254/255, 254/256, 254/257, 254/259, 255/256, 255/257, 255/258, 255/259, 256/257, 256/258, 256/259, 257/258, 257/259, 258/259, 323/330, 324/330, 325/330, 326/327, 326/328, 326/330, 327/328, 327/329, 327/330, 328/329, 328/330 or 329/330.

The DNA fragment of cpYFP is generated by PCR, phosphorus adding operation at the 5 'end is used for inactivation of the DNA fragment, pRSETb-amino acid binding protein linearization vectors containing different cleavage sites are generated by inverse PCR amplification, the linearized pRSETb-Atu2422 and cpYFP fragments phosphorylated at the 5' end are connected under the action of PEG4000 and T4DNA ligase to generate recombinant plasmids, the plates are subjected to a Kodak multifunctional living imaging system, clones with yellow fluorescence under the excitation of FITC channels are picked, and sequencing is completed by sea division on Beijing Hexahua major gene technologies Co.

After sequencing correctly, the recombinant plasmid was transformed into JM109 (DE 3) to induce expression, and the protein was purified and sized around 68kDa by SDS-PAGE. The size is consistent with the size of Atu2422-cpYFP fusion protein expressed by pRSETb-Atu2422-cpYFP and containing His-tag purification tag. The results are shown in FIG. 1.

Purified Atu2422-cpYFP fusion protein was subjected to alanine response screening, and the detection signal of the fusion fluorescent protein containing 100mM alanine was divided by the detection signal of the fusion fluorescent protein without alanine. As a result, as shown in FIG. 2, the detection results showed that the optical probes that responded more than 2-fold to alanine had optical probes that fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 (as shown in SEQ ID NO 10-15) or the corresponding amino acid positions of the family proteins thereof.

Example 3: expression and detection of cpGFP optical probes for different fusion sites

The amino acid green fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpGFP as in example 2. As shown in FIG. 3, the detection results showed that the optical probes that responded more than 2-fold to alanine had optical probes that fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins.

Example 4: expression and detection of cpBFP optical probes for different fusion sites

The amino acid blue fluorescent protein fluorescent probe was constructed by substituting cpYFP for cpBFP according to the method in example 2. As shown in FIG. 4, the detection results showed that the optical probes that responded more than 2-fold to alanine had optical probes that fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins.

Example 5: expression and detection of cpmAppe optical probes of different fusion sites

The amino acid red fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpmApple as in example 2. As shown in FIG. 5, the detection results showed that the optical probes that responded more than 2-fold to alanine had optical probes that fused at positions 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 or the corresponding amino acid positions of the family proteins.

Example 6: performance of optical probes

For the optical probes obtained in example 2 that responded more than 2 times to alanine, i.e.at 120/121, 121/122, 121/123,the 6 optical probes fused at positions 324/330, 325/330 and 326/330 were subjected to alanine detection in concentration gradient to detect the change of the ratio of the fluorescence intensity at 528nm emission at 420nm excitation and the fluorescence intensity at 528nm emission at 485nm excitation. K of 6 amino acid optical probes with fusion sites 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330 _d (binding constants) were 8.5mM, 0.02mM, 5.6mM, 0.13mM and 0.79mM, respectively, and the change amplitudes were 3.4-fold, 4.3-fold, 2.8-fold, 2.4-fold, 2.7-fold and 2.0-fold, respectively, and the results are shown in FIG. 6.

For the probe with fusion site 121/122 (Atu 2422-121/122-cpYFP), its specific detection of each amino acid was performed. Meanwhile, the specificity of the probe with the fusion site 121/122 to alanine was compared with the fusion protein obtained by fusing fluorescent protein cpYFP, cpGFP, cpBFP or cpmeAN_SNe to the N-terminal or C-terminal of Atu2422 as a control.

The results showed that the probe with fusion site 121/122 had a higher response to alanine, proline, valine, serine, isoleucine, threonine and cysteine, 4.3-fold, 4.5-fold, 4.0-fold, 3.2-fold, 2.4-fold and 3.0-fold, respectively, as shown in FIG. 7A. Meanwhile, fusion proteins obtained by fusing fluorescent proteins to the N-terminus or the C-terminus of Atu2422 were not responsive to alanine, whereas probes with fusion sites 121/122 were about 4.3-fold responsive to alanine, as shown in FIG. 7B.

Example 7: expression and detection of mutated cpYFP optical probes

The optical probe mutant was constructed on the basis of Atu 2422-121/122-cpYFP. The plasmid pRSETb-Atu2422-121/122-cpYFP is linearized by inverse PCR, the primer contains the base sequence of the desired mutation site, the obtained PCR product is connected by adding phosphorus under the action of PNK, T4DNA ligase and PEG4000, the site-specific saturation mutation plasmids of 8 sites of F77, A100, T102, D121, Y150, D226, G227 and Y275 are obtained, and sequencing is completed by Shanghai division of Beijing Liuhua macrogene technologies Co.

The results are shown in FIG. 8. Fluorescence detection showed that there were F77A, F77L, A100G, D121E, D121T, D121V, D226E, D226N, G227S and Y275F mutants responding to alanine more than 2-fold.

Example 8 Properties of optical Probe mutants

Illustratively, after subjecting purified amino acid optical probes Atu2422-121/122-cpYFP-G227S to 0mM and 500mM alanine treatment, respectively, for 10 minutes, fluorescence spectroscopy was performed using a fluorescence spectrophotometer.

Determination of excitation spectra: the excitation spectrum was recorded with an excitation range of 350nm to 510nm and an emission wavelength of 530nm, read every 1 nm. The results show that the probe has two excitation peaks at about 420 and 490nm, as shown in FIG. 9A.

Determination of emission spectra: the fixed excitation wavelengths were 420nm and 490nm, respectively, and the emission spectra were recorded at 505-600nm, read every 1 nm. The results show that the fluorescence intensity of the probe after addition of 500mM alanine was reduced by a factor of 0.78 with the addition of 0mM alanine at 420nm excitation; the fluorescence intensity at 490nm excitation was reduced by a factor of 0.22 with the addition of 0mM alanine. As shown in fig. 9B and 9C.

The purified Atu2422-121/122-cpYFP-G227S was subjected to alanine detection at a concentration gradient (0 to 100 mM). After 10 minutes of treatment of the purified probe, the change in the ratio of the fluorescence intensity at 528nm emission from 420nm excitation to the fluorescence intensity at 528nm emission from 485nm excitation was detected. The results are shown in FIG. 10, which shows the K of the amino acid optical probe _d (binding constant) was 2.8mM, and the change was 4-fold.

The Atu2422-121/122-cpYFP-G227S was tested for reactivity with 20 amino acids, and the results showed that it had good specificity, as shown in FIG. 11.

Example 9: subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

In this example, different localization signal peptides were used to fuse with the optical probe Atu2422-121/122-cpYFP-G227S to localize the optical probe to different organelles.

HeLa cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and fluorescence detected under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 12. The amino acid optical probe can be positioned in subcellular organelles including cytoplasm, mitochondria, nucleus, lysosome, endoplasmic reticulum, golgi apparatus and the like by fusing with different specific positioning signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.

HeLa cells were transfected with cytoplasmic expression optical probe plasmid for 36 hours, rinsed with PBS, and placed in HBSS solution to detect changes in the ratio of fluorescence intensity at 420nm excitation 528nm emission to fluorescence intensity at 485nm excitation 528nm emission over a 40min period. The results are shown in FIG. 13. 485/420 decreases gradually with the consumption of alanine. Alanine was then added and detection continued for 43min. The 485/420 of the alanine added sample gradually increased, while the 485/420 of the control group continued to decrease until unchanged.

Example 10: high throughput compound screening in living cells based on optical probes

In this example, we used HeLa cells expressing cytosolic expression Atu2422-121/122-cpYFP-G227S for high throughput compound screening.

Transfected HeLa cells were rinsed with PBS, placed in HBSS solution (without alanine) for 1 hour, and then treated with 10. Mu.M compound for 1 hour. Alanine was added dropwise to each sample. The change in the ratio of the fluorescence intensity at the 528nm emission of 420nm excitation to the fluorescence intensity at the 528nm emission of 485nm excitation was recorded using a microplate reader. Samples not treated with any compound were normalized as controls. The results are shown in FIG. 14. Of the 2000 compounds used, the vast majority of compounds had minimal effect on alanine entry into the cell. There are 11 compounds that increase the uptake of alanine by cells and 9 compounds that significantly decrease the uptake of alanine by cells.

Example 11: quantitative detection of alanine in blood by optical probe

In this example, purified Atu2422-121/122-cpYFP-G227S was used to analyze alanine in mouse and human blood supernatants.

After mixing Atu2422-121/122-cpYFP-G227S with the diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at the 528nm emission at 420nm excitation to the fluorescence intensity at the 528nm emission at 485nm excitation was detected using a microplate reader. As a result, as shown in FIG. 15, the alanine content in the blood of the mice was about 600. Mu.M, and the alanine content in the blood of the human was about 360. Mu.M.

The embodiment shows that the amino acid optical probe provided by the invention has relatively small molecular weight, is easy to mature, has large dynamic change of fluorescence and good specificity, can be expressed in cells by a gene operation method, and can be used for positioning and quantitatively detecting amino acids inside and outside the cells in real time; and enables high throughput compound screening.

Other embodiments

This specification describes a number of embodiments. It will be appreciated that various modifications may be made by those skilled in the art from a reading of this specification without departing from the spirit and scope of the invention, and are intended to be included within the scope of the appended claims.

Sequence listing

<110> university of Industy of Huadong

<120> amino acid optical probe, and preparation method and application thereof

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Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr

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Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe

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Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr

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Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser

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Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr

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Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr

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Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val

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Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu

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Pro Ser Lys Leu Gly His Lys Leu Asn

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Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Ser Glu Ile

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Lys Lys Gly Leu Arg Leu Lys Asp Gly Gly His Tyr Ala Ala Glu Val

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Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr

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Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr

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Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly

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Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Leu Val Ser Lys

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Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys

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Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly

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Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys

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Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro

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Gln Phe Met Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile

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Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg

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Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser

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Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr

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Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp

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Glu Ala

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Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

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Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Tyr Asn Ser Asp Asn Val Tyr Ile

115 120 125

Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg

130 135 140

His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln

145 150 155 160

Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

165 170 175

Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp

180 185 190

His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly

195 200 205

Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser

210 215 220

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

225 230 235 240

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu

245 250 255

Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr

260 265 270

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr

275 280 285

Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp

290 295 300

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

305 310 315 320

Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe

325 330 335

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

340 345 350

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 11

<211> 596

<212> PRT

<213> artificial sequence

<400> 11

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 12

<211> 595

<212> PRT

<213> artificial sequence

<400> 12

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gln

355 360 365

Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala

370 375 380

Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala

385 390 395 400

Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met

405 410 415

Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser

420 425 430

Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His

435 440 445

Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys

450 455 460

Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser

465 470 475 480

Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp

485 490 495

Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala

500 505 510

Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met

515 520 525

Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu

530 535 540

Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly

545 550 555 560

Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr Val

565 570 575

Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln

580 585 590

Gln Gly Ser

595

<210> 13

<211> 591

<212> PRT

<213> artificial sequence

<400> 13

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys

325 330 335

Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu

340 345 350

Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile

355 360 365

Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln

370 375 380

Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu

385 390 395 400

Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu

405 410 415

Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu

420 425 430

Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val

435 440 445

Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr

450 455 460

Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro

465 470 475 480

Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys

485 490 495

Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser

500 505 510

Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp

515 520 525

Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr

530 535 540

Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly

545 550 555 560

Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu Trp

565 570 575

Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 14

<211> 592

<212> PRT

<213> artificial sequence

<400> 14

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Pro Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp

325 330 335

Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val

340 345 350

Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro

355 360 365

Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe

370 375 380

Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val

385 390 395 400

Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu

405 410 415

Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu

420 425 430

Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp

435 440 445

Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala

450 455 460

Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu

465 470 475 480

Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys

485 490 495

Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys

500 505 510

Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys

515 520 525

Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp

530 535 540

Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp

545 550 555 560

Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr Glu

565 570 575

Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly Ser

580 585 590

<210> 15

<211> 593

<212> PRT

<213> artificial sequence

<400> 15

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Asp Gln Gln Gly Gly Ile Ala

115 120 125

Gly Lys Tyr Leu Ala Asp His Phe Lys Asp Ala Lys Val Ala Ile Ile

130 135 140

His Asp Lys Thr Pro Tyr Gly Gln Gly Leu Ala Asp Glu Thr Lys Lys

145 150 155 160

Ala Ala Asn Ala Ala Gly Val Thr Glu Val Met Tyr Glu Gly Val Asn

165 170 175

Val Gly Asp Lys Asp Phe Ser Ala Leu Ile Ser Lys Met Lys Glu Ala

180 185 190

Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu His Thr Glu Ala Gly Leu

195 200 205

Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu Lys Ala Lys Leu Val Ser

210 215 220

Gly Asp Gly Ile Val Ser Asn Glu Leu Ala Ser Ile Ala Gly Asp Ala

225 230 235 240

Val Glu Gly Thr Leu Asn Thr Phe Gly Pro Asp Pro Thr Leu Arg Pro

245 250 255

Glu Asn Lys Glu Leu Val Glu Lys Phe Lys Ala Ala Gly Phe Asn Pro

260 265 270

Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala Met Gln Ala Ile Ala Gly

275 280 285

Ala Ala Lys Ala Ala Gly Ser Val Glu Pro Glu Lys Val Ala Glu Ala

290 295 300

Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu Gly Glu Ile Ser Phe Asp

305 310 315 320

Glu Lys Gly Asp Pro Lys Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala

325 330 335

Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn

340 345 350

Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr

355 360 365

Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser

370 375 380

Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met

385 390 395 400

Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp

405 410 415

Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly

420 425 430

Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly

435 440 445

Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp

450 455 460

Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys

465 470 475 480

Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu

485 490 495

Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe

500 505 510

Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe

515 520 525

Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly

530 535 540

Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu

545 550 555 560

Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Val Met Tyr

565 570 575

Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile Gln Gln Gly

580 585 590

Ser

<210> 16

<211> 596

<212> PRT

<213> artificial sequence

<400> 16

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Ala Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 17

<211> 596

<212> PRT

<213> artificial sequence

<400> 17

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Leu Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 18

<211> 596

<212> PRT

<213> artificial sequence

<400> 18

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Gly Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 19

<211> 596

<212> PRT

<213> artificial sequence

<400> 19

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Glu Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 20

<211> 596

<212> PRT

<213> artificial sequence

<400> 20

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Thr Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 21

<211> 596

<212> PRT

<213> artificial sequence

<400> 21

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Val Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 22

<211> 596

<212> PRT

<213> artificial sequence

<400> 22

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Glu Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 23

<211> 596

<212> PRT

<213> artificial sequence

<400> 23

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asn Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 24

<211> 596

<212> PRT

<213> artificial sequence

<400> 24

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Ser Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Tyr Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 25

<211> 596

<212> PRT

<213> artificial sequence

<400> 25

Met Asp Val Val Ile Ala Val Gly Ala Pro Leu Thr Gly Pro Asn Ala

1 5 10 15

Ala Phe Gly Ala Gln Ile Gln Lys Gly Ala Glu Gln Ala Ala Lys Asp

20 25 30

Ile Asn Ala Ala Gly Gly Ile Asn Gly Glu Gln Ile Lys Ile Val Leu

35 40 45

Gly Asp Asp Val Ser Asp Pro Lys Gln Gly Ile Ser Val Ala Asn Lys

50 55 60

Phe Val Ala Asp Gly Val Lys Phe Val Val Gly His Phe Asn Ser Gly

65 70 75 80

Val Ser Ile Pro Ala Ser Glu Val Tyr Ala Glu Asn Gly Ile Leu Glu

85 90 95

Ile Thr Pro Ala Ala Thr Asn Pro Val Phe Thr Glu Arg Gly Leu Trp

100 105 110

Asn Thr Phe Arg Thr Cys Gly Arg Asp Tyr Asn Ser Asp Asn Val Tyr

115 120 125

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile

130 135 140

Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

145 150 155 160

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

165 170 175

Tyr Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg

180 185 190

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

195 200 205

Gly Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly

210 215 220

Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu

225 230 235 240

Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly

245 250 255

Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr

260 265 270

Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly

275 280 285

Tyr Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His

290 295 300

Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr

305 310 315 320

Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys

325 330 335

Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp

340 345 350

Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Asp

355 360 365

Gln Gln Gly Gly Ile Ala Gly Lys Tyr Leu Ala Asp His Phe Lys Asp

370 375 380

Ala Lys Val Ala Ile Ile His Asp Lys Thr Pro Tyr Gly Gln Gly Leu

385 390 395 400

Ala Asp Glu Thr Lys Lys Ala Ala Asn Ala Ala Gly Val Thr Glu Val

405 410 415

Met Tyr Glu Gly Val Asn Val Gly Asp Lys Asp Phe Ser Ala Leu Ile

420 425 430

Ser Lys Met Lys Glu Ala Gly Val Ser Ile Ile Tyr Trp Gly Gly Leu

435 440 445

His Thr Glu Ala Gly Leu Ile Ile Arg Gln Ala Ala Asp Gln Gly Leu

450 455 460

Lys Ala Lys Leu Val Ser Gly Asp Gly Ile Val Ser Asn Glu Leu Ala

465 470 475 480

Ser Ile Ala Gly Asp Ala Val Glu Gly Thr Leu Asn Thr Phe Gly Pro

485 490 495

Asp Pro Thr Leu Arg Pro Glu Asn Lys Glu Leu Val Glu Lys Phe Lys

500 505 510

Ala Ala Gly Phe Asn Pro Glu Ala Phe Thr Leu Tyr Ser Tyr Ala Ala

515 520 525

Met Gln Ala Ile Ala Gly Ala Ala Lys Ala Ala Gly Ser Val Glu Pro

530 535 540

Glu Lys Val Ala Glu Ala Leu Lys Lys Gly Ser Phe Pro Thr Ala Leu

545 550 555 560

Gly Glu Ile Ser Phe Asp Glu Lys Gly Asp Pro Lys Leu Pro Gly Tyr

565 570 575

Val Met Tyr Glu Trp Lys Lys Gly Pro Asp Gly Lys Phe Thr Tyr Ile

580 585 590

Gln Gln Gly Ser

595

<210> 26

<211> 1791

<212> DNA

<213> artificial sequence

<400> 26

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcgacggta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

<210> 27

<211> 1791

<212> DNA

<213> artificial sequence

<400> 27

atggatgtcg tgatcgctgt cggcgcaccg ctgaccggcc cgaacgctgc tttcggcgct 60

cagatccaga agggtgccga acaggctgcg aaagacatca atgctgccgg cggtatcaat 120

ggcgagcaga ttaagatcgt gctgggcgac gacgtatccg accccaagca gggtatttcg 180

gttgccaaca aattcgttgc tgacggcgtg aaattcgttg tcggtcactt caactcgggt 240

gtttccattc cggcatcgga agtttatgcc gaaaacggca ttctcgaaat cacgcccgct 300

gcgaccaacc cggtctttac cgagcgtggc ctgtggaaca ccttccgcac ctgcggccgt 360

gactacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 420

aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 480

cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 540

cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 600

gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 660

ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 720

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 780

acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 840

cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 900

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 960

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1020

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1080

gggcacaagc tggagtacaa cgaccagcag ggcggcattg ccggcaagta cctggccgat 1140

catttcaagg acgccaaggt cgccatcatt cacgacaaga cgccttatgg tcagggtctt 1200

gccgatgaaa ccaaaaaggc tgccaatgct gccggcgtga ctgaggtcat gtatgaaggc 1260

gtcaacgtcg gcgacaagga cttctccgcg ctgatctcga agatgaagga agccggcgtt 1320

tccatcatct actggggcgg cctgcacacc gaagccggcc tgatcatccg ccaggcggct 1380

gaccagggtc tgaaggccaa gctcgtttcg ggcgactcta ttgtctcgaa cgaacttgct 1440

tccatcgccg gcgacgccgt cgagggcacg ctgaacacct tcggccctga tccgacgctg 1500

cgcccggaaa acaaggaact ggtagagaag ttcaaggccg ccggcttcaa cccggaagcc 1560

tacacgctct actcctatgc cgcgatgcag gcgattgcag gcgcagcgaa ggctgcgggt 1620

tccgtggagc cggaaaaggt tgccgaagcc ctgaagaagg gctccttccc gaccgcactc 1680

ggcgaaatct ccttcgatga gaagggcgac ccgaagcttc ccggctacgt catgtacgaa 1740

tggaagaagg gtccggacgg caagttcacc tacatccagc agggcagcta a 1791

Claims

1. An optical probe comprising an amino acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the amino acid-sensitive polypeptide, the sequence of the amino acid-sensitive polypeptide is shown in SEQ ID No. 1 or as SEQ ID No. 1 and has one or more mutations selected from the group consisting of: F77A, F77Q, F77L, A100G, D121E, D121T, D121V, Y150S, D226E, D226N, G227S, Y275Q and Y275F,

the optically active polypeptide is located at one or more sites of the amino acid-sensitive polypeptide selected from the group consisting of: 120/121, 121/122, 121/123, 324/330, 325/330 and 326/330.

2. The optical probe of claim 1, wherein the optically active polypeptide is a fluorescent protein.

3. The optical probe of claim 1, wherein the optical probe is set forth in any one of SEQ ID NOs 10 to 25.

4. A nucleic acid molecule comprising a nucleic acid sequence encoding the optical probe of any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4 operably linked to an expression control sequence.

6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A method of preparing the optical probe of any one of claims 1-3, comprising the steps of:

(1) Transferring the expression vector of claim 5 into a host cell,

(2) Culturing said host cell under conditions suitable for expression of said expression vector, and

(3) Isolating the optical probe from the host cell.

8. Use of an optical probe according to any one of claims 1-3 for the preparation of a kit for detecting alanine in a sample.

9. A test kit comprising the optical probe of any one of claims 1-3 or the optical probe prepared by the method of claim 7.

10. A non-diagnostic method for detecting alanine in a sample, comprising: contacting the optical probe of any one of claims 1-3 with a sample, detecting a change in an optically active polypeptide, and detecting alanine in the sample based on the change in the optically active polypeptide.

11. A method of screening a compound comprising: contacting a candidate compound with a cell expressing an optical probe according to any one of claims 1-3, determining the change in the optically active polypeptide, and screening for a compound that affects a change in alanine content based on the change in the optically active polypeptide.

12. Use of an optical probe comprising an amino acid sensitive polypeptide and an optically active polypeptide for the preparation of a kit for detecting an amino acid in a sample, said amino acid being selected from one or more of valine, serine, isoleucine, threonine and cysteine,

the optically active polypeptide is located within the sequence of the amino acid-sensitive polypeptide,

the sequence of the amino acid sensitive polypeptide is shown as SEQ ID NO. 1,

the optically active polypeptide is located at position 121/122 of the amino acid-sensitive polypeptide.

13. A non-diagnostic method for detecting an amino acid in a sample comprising: contacting an optical probe comprising an amino acid-sensitive polypeptide and an optically active polypeptide with a sample, detecting a change in the optically active polypeptide, and detecting an amino acid in the sample based on the change in the optically active polypeptide, the amino acid selected from one or more of valine, serine, isoleucine, threonine, and cysteine,

the sequence of the amino acid sensitive polypeptide is shown as SEQ ID NO. 1,

14. A method of screening a compound comprising: contacting a candidate compound with a cell expressing an optical probe comprising an amino acid-sensitive polypeptide and an optically active polypeptide, determining the change in the optically active polypeptide, and screening for a compound having an effect on the change in the content of an amino acid selected from one or more of valine, serine, isoleucine, threonine and cysteine according to the change in the optically active polypeptide,

the sequence of the amino acid sensitive polypeptide is shown as SEQ ID NO. 1,