CN114703168B - Heparinase III - Google Patents
Heparinase III Download PDFInfo
- Publication number
- CN114703168B CN114703168B CN202210203224.7A CN202210203224A CN114703168B CN 114703168 B CN114703168 B CN 114703168B CN 202210203224 A CN202210203224 A CN 202210203224A CN 114703168 B CN114703168 B CN 114703168B
- Authority
- CN
- China
- Prior art keywords
- ala
- lys
- asn
- leu
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02008—Heparin-sulfate lyase (4.2.2.8)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides heparinase III, a coding nucleotide sequence thereof, a recombinant vector and a host cell comprising the nucleotide sequence and application thereof, and belongs to the fields of genetic engineering and fermentation engineering. The heparanase III comprises an amino acid sequence shown as a Seq ID No.2 or a Seq ID No.3, and compared with the original heparanase III, the modified heparanase III has the advantages that the enzyme activity is not reduced, and the stability is enhanced.
Description
Technical Field
The invention relates to the fields of genetic engineering and fermentation engineering, in particular to heparinase III, a coding nucleotide sequence thereof, a recombinant vector comprising the nucleotide sequence, a host cell and application thereof.
Background
Heparinase (heparinase) is a class of polysaccharide-cleaving enzymes acting on heparin (heparin) or heparan sulfate (heparan sulfate), and is present in a variety of microorganisms, with heparinase from Flavobacterium heparinum being the most common. There are only three heparanases from Flavobacterium heparinum, heparinase I (EC 4.2.2.7), heparinase II (No EC code) and heparinase III (EC 4.2.2.8), respectively (Robert J. Linhardt et al purification and characterization of heparin lyases from Flavobacterium heparinum JBC 1992Vol. 267:24347-24355).
Heparanase III acts mainly on heparan sulphate with a molecular weight of 73kDa. In contrast to the other two heparanases, studies have shown that heparinase III acts on heparinoids in the extracellular matrix, producing active heparin small molecules that inhibit proliferation of capillary epithelial cells, thereby inhibiting growth of tumor cells and reducing metastasis and spread of cancer cells (Liu DF, prjasek K, sri ver Z, et al, heparinase III and uses thereof: united States, U.S. Pat. No.4,6869789B 2[ P ]. 205-5-22).
Disclosure of Invention
At present, the stability of the heparanase III is poor, and the heparanase III which improves the enzyme stability on the basis of ensuring the enzyme catalytic activity is seldom researched and developed through genetic engineering means.
In view of the above drawbacks, it is an object of the present invention to provide heparinase III and its coding nucleotide sequences, recombinant vectors and host cells comprising the nucleotide sequences.
The invention provides a heparanase III, which comprises an amino acid sequence shown as a Seq ID No.2 or a Seq ID No. 3.
The original amino acid sequence of heparanase III is shown as Seq ID No. 1.
Seq ID No.2 shows that the amino acid sequence is that the Q glutamine at positions 85, 114, 403 and 547 in Seq ID No.1 is replaced by A alanine.
The amino acid sequence of Seq ID No.3 is represented by the substitution of Q glutamine at position 403 with V valine and the substitution of Q glutamine at positions 85, 114 and 547 with A alanine in Seq ID No. 1.
It is another object of the present invention to provide a nucleotide sequence encoding heparanase III as described above.
Further, the nucleotide sequence is shown as Seq ID No.4 or Seq ID No.5, wherein Seq ID No.4 is the nucleotide sequence encoding heparinase III of Seq ID No.2 and Seq ID No.5 is the nucleotide sequence encoding heparinase III of Seq ID No. 3.
It is a further object of the present invention to provide a recombinant vector comprising at least one of the above nucleotide sequences.
Further, the recombinant vector is a eukaryotic recombinant vector.
Further, the eukaryotic cell recombinant vector is any one of pPink-HC, pPICZaA and pPICZ A.
Preferably, the eukaryotic recombinant vector is pPink-HC.
It is a further object of the present invention to provide a host cell comprising any of the recombinant vectors described above.
Further, the host cell is pichia pastoris.
Still further, the pichia pastoris is X33.
The invention also provides a preparation method of heparinase III, which comprises the following steps:
(a) Synthesis of nucleotide sequences encoding heparanase III described above
(b) Combining the nucleotide sequence in (a) with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;
(c) Introducing the recombinant vector in (b) into a host cell, culturing and inducing expression, and purifying to obtain the heparanase III.
Preferably, step (c) uses Buffer W to purify the Ssep-Tactin column.
Compared with the prior art, the invention has the beneficial effects that:
the invention modifies protease enzyme cutting sites which possibly influence the stability of the heparanase III in the amino acid sequence (shown as Seq ID No. 1) of the prior heparanase III, specifically, the Q glutamine at 85, 114, 403 and 547 positions in the amino acid sequence of the prior heparanase III is subjected to site-directed mutation to A alanine or V valine, so that the stability, especially the heat stability, the enzyme activity half-life and the enzyme activity half-life of the heparanase III can be improved, when the 85, 114, 403 and 547 positions in the original amino acid sequence are replaced by A alanine, the stability is improved obviously, the enzyme activity half-life at the optimal reaction temperature of 30 ℃ reaches about 47 hours, when the 85, 114 and 547 positions in the original amino acid sequence are replaced by A alanine and the 403 position is replaced by V valine, the stability is improved obviously, and the enzyme activity half-life at the optimal reaction temperature of 30 ℃ is longer.
The nucleotide sequence provided by the invention is used for encoding the heparanase III, the DNA sequence is optimized according to the codon usage preference of pichia pastoris by adopting a rare codon optimization method, and the expression efficiency of the optimized sequence in the pichia pastoris is obviously improved.
The recombinant vector and host cell provided by the invention comprise the nucleotide sequence and can be used for expressing the heparinase III.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the following description will briefly explain the drawings needed in the embodiments or the prior art description, and it is obvious that the drawings in the following description are some embodiments of the invention and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an SDS-PAGE electrophoresis chart of example 8 of the present invention
FIG. 2 is a graph showing the stability measurement of heparinase III of example 10 of the present invention before and after modification.
Detailed Description
"amino acid" refers to any monomeric unit that may be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 naturally or genetically encoded α -amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). In the case where "X" residues are undefined, these should be defined as "any amino acid". The structure of these 20 natural amino acids is shown, for example, in Stryer et al, bioChemistry, 5 th edition, freeman and Company (202). Additional amino acids such as Selenocysteine and pyrrolysine can also be genetically encoded (Stadtman (1996) "Selenocysteine," Annu Rev biochem.65:83-100 and Ibba et al (202) "Genetic code: introducing pyrrolysine," Curr biol.12 (13): R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., with modified side chains and/or backbones), and amino acid analogs. (see, e.g., zhang et al (204), "Selectiveincorporation of-hydroxytryptophan into Proteins in mammalian cells," proc. Natl. Acad. Sci. U.S.A.11 (24): 8882-8887, anderson et al (204) "An expandedgenetic code with a functional quadruplet codon" Proc.Natl.Acad.Sci.U.S.A.11 (20): 7566-75171, ikeda et al (203) "Synthesis of a novel histidineanalogue and its efficient incorporation into a Protein in vivo," ProteinEng. Des. Sel.16 (9): 699-706, "Chin et al (203)" An Expanded Eukaryotic GeneticCode, "Science 31 (5635): 964-967," james et al (201) "Kinetic characterizationof ribonuclease S mutants containing photoisomer-izable phenylazophenylalanineresidues," Protein Eng. Des. Sel.14 (12): 983-991, "Kohrer et al (201)" Importof amber and ochre suppressor tRNAs into mammalian cells: A general approachto site-specific insertion of amino acid analogues into Proteins, "Proc. Natl. Acad. Sci. A.98 (25): 14310-14315," Bacher et al (201) "Selection andCharacterization of Escherichia coli Variants Capable of Growth on anOtherwise Toxic Tryptophan Analogue," J. Bacterol. 183 (18): 5414-A Mutant Escherichia coli Tyrosyl, "Hamano-Takaku et al (2000)," J. Biol. Chem.275 (51): 4324-4328, "Budisco et al (201)" Protein { beta- (26) } (25-1291), "Proc. Natl. Acad. Scuter. A.98 (25):.1431-1292).
To further illustrate, the amino acid is typically an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, mercapto, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxy, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, phospho, phosphonyl, phosphine, heterocycle, ketene, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photosensitive cross-linker, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids containing new functional groups, amino acids that interact covalently or non-covalently with other molecules, photolabile (photocaged) and/or photoisomerisable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogues, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids comprising carbon linked sugars, redox active amino acids, amino acids comprising amino thio acids, and amino acids comprising one or more toxic moieties.
The original amino acid sequence of heparanase III in the present invention (shown as Seq ID No. 1) is a sequence derived from heparanase III already published in NCBI database.
Then, on the basis of the amino acid sequence, according to the preference of the expression host cell for codon usage, the nucleotide sequence corresponding to the amino acid sequence is subjected to codon optimization so as to improve the translation efficiency of the target sequence in the fermentation process of the host cell, so that more target proteins can be obtained as much as possible.
After the target sequence is synthesized by a target company, the mutation of target amino acid is carried out on the basis of the target sequence, and the fragments are connected through Gibson reaction after the fragment PCR amplification product to form the mutated vector.
The polymerase chain reaction (polymerase chain reaction, PCR) uses a piece of DNA as a template that is amplified to a sufficient amount in the presence of a DNA polymerase and nucleotide substrates for structural and functional analysis. The PCR detection method has extremely important significance in the aspects of rapidly diagnosing bacterial infectious diseases clinically and the like.
The principle of PCR is used to amplify DNA fragments between two known sequences, similar to the replication process of natural DNA. The target DNA fragment can be amplified by taking the DNA molecule to be amplified as a template, a pair of oligonucleotide fragments respectively complementary with the 5 '-end and the 3' -end of the template as primers, extending along the template chain according to a semi-reserved replication mechanism under the action of DNA polymerase until new DNA synthesis is completed, and repeating the process.
Gibson assembly was first proposed in 2009 by Daniel Gibson doctor and his colleague J.Craig Venter. Gibson assembly is well suited for splicing multiple linear DNA fragments, as well as for inserting the target DNA into a vector. Firstly, it is necessary to add a homologous fragment (added by PCR) to the end of the DNA fragment; then, these DNA fragments were mixed with a Master Mix (containing three enzymes) and incubated for one hour.
Wherein the Master Mix contains three different types of enzymes:
an exonuclease digests DNA starting from the 5' end, producing a growing cohesive end, which facilitates mating with an otherwise homologous end;
a polymerase for repairing a gap;
a DNA ligase, which performs traceless splicing to form a complete DNA molecule.
Designing a primer near a mutant amino acid site, changing an amino acid codon to be contained in the primer, connecting adjacent fragments to a homologous sequence required by connection of adjacent fragments in the sequence, purifying the PCR product after amplification, and connecting the PCR amplified fragments by using a Gibson reaction reagent to obtain a heparinase III recombinant expression vector sequence with the amino acid mutation at a designated position.
Finally, the optimized amino acid sequence shown as the Seq ID No.2 or the Seq ID No.3 is obtained. In addition, seq ID No.2 indicates that the amino acid sequence is such that all of the Q glutamine at positions 85, 114, 403, and 547 in Seq ID No.1 is replaced with A alanine. The amino acid sequence of Seq ID No.3 is represented by the substitution of Q glutamine at position 403 with V valine and the substitution of Q glutamine at positions 85, 114 and 547 with A alanine in Seq ID No. 1.
Coding sequence: the term "coding sequence" as used herein includes nucleotide sequences which directly indicate the amino acid sequence of its protein product. The boundaries of the coding sequence are typically defined by an open reading frame, which typically begins with the ATG start codon. The coding sequence generally includes DNA, cDNA and recombinant nucleotide sequences.
The term "nucleotide", in addition to referring to naturally occurring ribonucleotides or deoxyribonucleotide monomers, is also understood herein to refer to structural variants thereof, including derivatives and analogs, which are functionally equivalent with respect to the particular context in which the nucleotide is used, unless the context clearly indicates otherwise.
The terms "codon-optimized", "codon-optimized" or "codon usage preference" refer to the practice of selecting codons (i.e., codon usage) in such a way that expression is optimized or customized as desired (i.e., techniques to improve protein expression in an organism by increasing the translational efficiency of the gene of interest). In other words, codon optimization is a method of adjusting codons to match host tRNA abundance and has traditionally been used to express heterologous genes. New strategies for optimizing heterologous expression consider global nucleotide content, such as local mRNA folding, codon pair bias, codon ramp (codon ramp), or codon correlation. Codon optimisation is possible because of the degeneracy of the codons. Degeneracy results because there are more codons than can encode amino acids. Thus, the vast majority of amino acids are encoded by multiple codons, meaning that there are multiple tRNAs (with different anti-codon loops) carrying any given amino acid. Thus, different codons can be used without altering the encoded amino acid sequence. That is, the gene or fragment of the nucleic acid may be mutated/altered (or synthesized de novo) to alter the codon encoding a particular amino acid without altering the amino acid sequence of the polypeptide/protein itself. For example, rare codons may be replaced with richer codons while leaving the amino acid sequence unchanged.
The term "host cell" refers to single-cell prokaryotes and eukaryote organisms (e.g., bacteria, yeast, and actinomycetes) that are single cells from higher plants or animals when grown in cell culture. The "host cell" may be an animal host cell, a plant host cell, a yeast host cell, a fungal host cell, a protozoan host cell, and a prokaryotic host cell.
For example, the host cell is selected from: pichia pastoris (Pichia pastoris), pichia angusta (Pichia angusta) (Hansenula polymorpha (Hansenula polymorpha)), pichia finland (Pichia finlandica), pichia pastoris (Pichia trehalophila), pichia koclama, pichia pastoris (Pichia membranaefaciens), pichia minuta (Ogataea minista, pichia lindneri), pichia pastoris (Aspergillus niger), pichia pastoris (Pichia pastoris), pichia thermotolerans (Pichia thermotolerans), liu Bichi yeast (Pichia salictaria), pichia oaca (Pichia guebias), pichia pastoris (Pichia pastoris), pichia stipitis (Pichia methanolica), pichia lipolytica (Yarrowia Lipolytica), kluyveromyces lactis (Kluyveromyces lactis), zygosaccharomyces rouxii (Zygosaccharomyces rouxii), zygosaccharomyces bailii (Zygosaccharomyces bailii), wawangear (Schwanniomyces occidentalis), kluyveromyces (Aspergillus niger), zygosaccharomyces niger (5223), aspergillus kawakava (793), aspergillus flavus (6368), aspergillus flavus (Candida pu1), candida pu1 (Candida utilis), candida oxydani (Candida oxydans (Candida utilis), candida glabrata (Candida) and Aspergillus flava (Candida utilis) Debaryomyces hansenii (Debaromyces hansenii) and Saccharomyces cerevisiae. In one embodiment of the invention, the host cell may be Pichia or Saccharomyces cerevisiae. In one embodiment of the invention, the host cell is Saccharomyces cerevisiae (Saccharomyces cerevisiae). In a preferred embodiment of the invention, the host cell is Pichia (Pichia), and a Pichia pastoris (Pichia pastoris) expression system is used. Compared with a prokaryotic expression system, the yeast which is a single-cell eukaryote has the following characteristics: first, yeast is eukaryotic, which can make glycosylation of certain proteins more stable, and post-translational modifications such as correct disulfide bond formation and removal of signal peptides, N-binding and O-binding glycosylation modifications; secondly, the yeast has a single-cell microorganism structure, and has the advantages of rapid growth of a bacterial system, easiness in genetic engineering operation and the like; compared with E.coli, yeast has no endotoxin, no lysogenic viruses, and is closely related to human beings, and is non-pathogenic, and has long been used in the bread and wine industry. Yeast is also an ideal secretory expression system. Normally, no protein is added in the yeast culture medium, and the normal yeast secreted protein is only 0.5% of the total protein of the yeast cells; the produced exogenous protein can be secreted into the culture medium, so that the separation and purification of the product are facilitated, and the cost can be reduced to a great extent. Proteins that require posttranslational proper folding, natural secretion, and intracellular instability or toxicity are well suited for expression in such secretory expression systems.
The pichia pastoris system has several advantages: (1) The system adopts a methanol-induced promoter, and a single copy of ethanol oxidase gene can generate 30% ethanol oxidase of the total soluble protein amount of cells under the drive of the promoter, and the promoter is introduced into an expression vector to drive the expression of exogenous genes. (2) In the 70s, pichia pastoris is used for preparing single cell proteins, so that the fermentation technology of pichia pastoris is produced, and 100 g of stem cells per liter of culture can be achieved. (3) High level secretion in protein-free medium, the expression vector carries Saccharomyces cerevisiae factor a signal peptide, and can secrete the expression product to outside. The expression medium comprises inorganic salts, trace elements, biotin and a carbon source, and is free of toxins and pyrogens, so that the secreted expression product is very easy to purify. (4) The glycosylated form of Pichia pastoris is more mammalian, the expression product N-bound mannose is generally only (8-14) in number, and as much as (50-150) in Saccharomyces cerevisiae. Moreover, unlike Saccharomyces cerevisiae which contains a combination of terminal a-1, 3-mannose, this modification results in an immune heritability. (5) The plasmid stability is good, the Pichia pastoris has no plasmid, allows the tandem repeat of the expression component, and is suitable for integrating to chromosome to express exogenous genes. (6) The secretion product has good permeability, and the excessive glycosylation in Saccharomyces cerevisiae causes a plurality of mannose residues on each N-binding oligosaccharide chain, thereby further influencing the permeability. In general, exogenous proteins with molecular weight of more than 20kDa cannot be secreted into the culture solution, but Pichia pastoris does not have the phenomenon, and the Pichia pastoris has a set of secretion ways which are very similar to eukaryotes in vivo and are secreted from an endoplasmic reticulum to the outside through a Golgi apparatus and vesicles. Both the steering enzyme with the molecular weight of more than 50kDa and the human serum protein can be secreted outside cells, and the expression quantity is at the level of 1 g/L.
In a preferred embodiment of the present invention, the nucleotide sequence corresponding to the above amino acid sequence is codon optimized according to the preference of expression Pichia pastoris for codon usage, and the protein sequence of heparinase III is entered into a codon preference analysis software, for example Nanjing Jinsi Biotechnology LimitedGenSmart developed by Severe TM Codon Optimization sequence optimization software performs sequence optimization (https:// www.genscript.com/tools/genetic-code-optimization); under the premise of ensuring that the protein sequence of heparinase III is unchanged and only utilizing the degeneracy of codons, codons which are low in use frequency and can influence ribosome passing efficiency in the translation process in Pichia pastoris are replaced by codons with high use frequency, so that a nucleotide sequence after codon optimization is obtained, as shown in a Seq ID No.4 or a Seq ID No.5 in a sequence table. Wherein, seq ID No.4 is the nucleotide sequence encoding heparinase III described by Seq ID No.2, and Seq ID No.5 is the nucleotide sequence encoding heparinase III described by Seq ID No. 3.
Expression: the term "expression" in this context includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.
The term "vector" refers to a piece of DNA, typically double stranded, into which a piece of exogenous DNA may have been inserted. The vector may be, for example, of plasmid origin. The vector contains a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector in a host cell. Exogenous DNA is defined as heterologous DNA, which is DNA that is not found naturally in the host cell, e.g., that replicates a vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Vectors are used to transport exogenous or heterologous DNA into a suitable host cell. Once in the host cell, the vector may replicate independently of or simultaneously with the host chromosomal DNA, and several copies of the vector and its inserted DNA may be produced. In addition, the vector may also contain the necessary elements to allow transcription of the inserted DNA into mRNA molecules or otherwise cause replication of the inserted DNA into multiple copies of RNA. Some expression vectors additionally contain sequence elements near the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Thus, many molecules of mRNA and polypeptide encoded by the inserted DNA can be rapidly synthesized.
Expression vector: the term "expression vector" in this context includes a linear or circular DNA molecule comprising a fragment encoding a polypeptide of the invention, and which fragment may be operably linked to other fragments that allow for its transcription.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently treated by recombinant DNA methods and which can express the nucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may comprise any means (means) to ensure self-replication. Alternatively, the vector may be integrated into the genome and replicated together with the chromosome(s) into which it has been integrated upon introduction into the host cell. Alternatively, a single vector or plasmid, or two or more vectors or plasmids containing in total all the DNA to be introduced into the genome of the host cell, or transposons may be used.
In one embodiment of the invention, the recombinant vector comprises a nucleotide sequence encoding the amino acid sequence shown in Seq ID No.2 or Seq ID No.3, preferably, when the host cell is selected from Pichia pastoris, the recombinant vector comprises the nucleotide sequence shown in Seq ID No.4 or Seq ID No. 5.
In one embodiment of the invention, the recombinant vector is a eukaryotic recombinant vector. The expression vector adopted by the recombinant vector can be pPink-HC, pPICZaA, pPICZ A, pPICZ, pPICZ alpha, pGAPZ, pGAPZ, pHBM A, pPIC9K, pPIC9K-His, pPIC3.5K, pPIC9, pPICZ alpha A, pAO815, pPIC9K-His, pHIL-S1, pGADT7, pGBKT7, pWB980, pT3 and the like; preference is given to pPink-HC, pPICZaA and pPICZ A, particular preference to pPink-HC. The above-mentioned vectors are all commercially available.
In a preferred embodiment of the invention, the eukaryotic recombinant vector is pPink-HC. In another preferred embodiment of the invention, the eukaryotic recombinant vector is pPICZaA.
In another preferred embodiment of the invention, the eukaryotic recombinant vector is pPICZ a.
In one embodiment of the invention, the nucleotide sequence corresponding to the amino acid sequence is codon optimized according to the preference of pichia pastoris for codon usage, and the protein sequence of heparinase III is input into GenSmart which is independently developed by Nanjing Jinsri Biotechnology Co., ltd TM Codon Optimization sequence optimization software performs sequence optimization (https:// www.genscript.com/tools/genetic-code-optimization); under the premise of ensuring that the protein sequence of heparinase III is unchanged and only utilizing the degeneracy of codons, codons which are low in use frequency and can influence ribosome passing efficiency in the translation process in Pichia pastoris are replaced by codons with high use frequency, so that the nucleotide sequence of the amino acid sequence shown by the coding Seq ID No.2 or the Seq ID No.3 is obtained.
In a preferred embodiment of the invention, the codon-optimized nucleotide sequence is the nucleotide sequence shown in Seq ID No.4 or Seq ID No. 5.
In one embodiment of the invention, the above nucleotide sequence is combined with a eukaryotic recombinant vector to obtain a recombinant vector. For example, the above nucleotide sequence is combined with pPink-HC to obtain a pPink-HC recombinant vector.
In one embodiment of the invention, the recombinant vector described above is introduced into a host cell, such as Pichia pastoris.
In one embodiment of the invention, the above nucleotide sequence is combined with pPICZaA to obtain a pPICZaA recombinant vector.
In one embodiment of the invention, the recombinant vector described above is introduced into a host cell, such as piczaa recombinant vector into pichia pastoris.
In one embodiment of the invention, the above nucleotide sequence is combined with pPICZ a to obtain a pPICZ a recombinant vector.
In one embodiment of the invention, the above recombinant vector is introduced into a host cell, such as picz a recombinant vector into pichia pastoris.
Means for purifying recombinant proteins are well known in the art and may include clarification (e.g., filtration or centrifugation), affinity chromatography, immunoaffinity chromatography, protein a (or G) chromatography, ion exchange (i.e., cationic and/or anionic) chromatography, size exclusion chromatography, adsorption chromatography, hydrophobic interaction chromatography, reverse phase chromatography, ultracentrifugation, precipitation, immunoprecipitation, extraction, phase separation, and the like.
The methods described herein comprise expressing a recombinant protein in a host cell line engineered to have at least one labeled Host Cell Protein (HCP), wherein the at least one labeled HCP is labeled with at least one purification tag. Typically, the labeled HCP is a protein that is highly abundant, difficult to remove during downstream purification processes, and/or affects product quality (e.g., residual proteases may degrade the biologic therapeutic product, thereby reducing its efficacy). HCPs having these features are referred to as "problematic" HCPs. Typically, the labeled HCP is a protein that is essential for cell survival and/or cell function (and thus, is not a good candidate for a gene knockout strategy). They may be captured in a separation process (e.g., a separation column). Non-limiting embodiments of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin binding domains (CBD tags), maltose binding proteins (MBP tags), flag tags, strep tags, and variants thereof (e.g., strep tags) and His tags.
In some embodiments, the disclosed polypeptides comprise Strep tags, e.g., strep tags. Adding strep II tag sequence at C end of heparinase III, purifying cell crude extract by desulfurizing biotin purifying column and using Buffer W to purify Srep-Tactin column, and purifying target protein heparinase III by means of interaction between strep II tag and biotin.
In one embodiment of the invention, the crushing pretreatment is carried out prior to purification, in particular as follows:
(a) Taking the thallus fermentation liquor of induced expression for low-temperature centrifugation;
(b) Collecting supernatant, and suspending by using Buffer W;
(c) Sonicating cells on an ice-water mixture;
(d) The suspension was centrifuged.
Preferably, the lysate is viscous, and RNase A (10 ug/ml) and Dnase I (5 ug/ml) may be added and incubated on ice.
In one embodiment of the invention, the heparanase III purification steps are as follows:
(a) Cleaning a Srep-Tactin column by using Buffer W, and then carrying out column loading operation;
(b) Washing the column by using Buffer W, and collecting eluent;
(c) Buffer E was added and the eluate collected.
(d) The D-sulfate biotin is removed by Buffer R in several times, and after complete removal, the column is washed with Buffer W.
Examples
The invention is further described below in conjunction with the detailed description. It should be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the contents of the present invention, and such equivalents are also within the scope of the present invention as defined in the appended claims.
Materials, reagents and the like used in the examples described below were commercially available unless otherwise specified. In the following examples, conventional methods are used unless otherwise specified. The DNA sequence optimization was done by the biotechnology company Jin Weizhi, su. (Pichia pastoris available from Thermo Fisher company under the product number A11152.Pichia Pink) TM The carrier kit was purchased from Thermo Fisher company under product number a11152.pPink-HC was purchased from Thermo Fisher, inc., product No. A11152 (expression vector, strain, both included in the kit).
EXAMPLE 1 Synthesis and screening of amino acid III sequences
The original amino acid sequence of heparanase III is the sequence from heparanase III already published in the NCBI database.
Then, on the basis of the amino acid sequence, according to the preference of the expression host Pichia pastoris for codon usage, the nucleotide sequence corresponding to the amino acid sequence is subjected to codon optimization, so as to improve the translation efficiency of the target sequence in the fermentation process of Pichia pastoris, and further obtain more target proteins as much as possible.
After the target sequence is synthesized by the Suzhou Jin Weizhi biotechnology company, the mutation of target amino acid is carried out on the basis of the target sequence, and the fragments are connected to form a mutated vector through Gibson reaction after the fragment PCR amplification product.
Designing a primer near a mutant amino acid site, changing an amino acid codon to be contained in the primer, connecting adjacent fragments to a homologous sequence required by connection of adjacent fragments in the sequence, purifying the PCR product after amplification, and connecting the PCR amplified fragments by using a Gibson reaction reagent to obtain a heparinase III recombinant expression vector sequence with the amino acid mutation at a designated position.
The amino acid sequence shown as Seq ID No.2 or Seq ID No.3 was obtained.
EXAMPLE 2 Synthesis of heparanase III nucleotide sequence and construction of recombinant expression vector
(a) The coding region sequence of heparinase III from Flavobacterium heparinum contains a large number of rare codons, which affect the protein translation efficiency when expressed by using Pichia pastoris as a host cell, so that codon optimization is performed. The protein sequence of heparinase III is entered into codon preference analysis software, and GenSmart developed by Nanjing Jinsri biotechnology Co., ltd TM Codon Optimization sequence optimization software performs sequence optimization (https:// www.genscript.com/tools/genetic-code-optimization);
(b) Optimizing the coding region sequence of the preliminary screened heparanase III in the example 1 by using the software according to the codon usage preference of pichia pastoris in a pichia pastoris codon usage preference data table, and replacing codons which have lower usage frequency and can influence the ribosome passing efficiency in the translation process by codons with higher usage frequency under the premise of ensuring that the protein sequence of the heparanase III is unchanged and only utilizing the degeneracy of codons to obtain a nucleotide sequence after codon optimization, wherein the obtained sequence is shown as a Seq ID No.4 or a Seq ID No.5 in a sequence table;
and then connecting the obtained codon-optimized nucleotide sequence of the heparanase III with a pPink-HC eukaryotic cell expression vector by a conventional molecular biological means to construct a pPink-HC-heparanase III recombinant expression vector.
Example 3 eukaryotic cell expression vector was pPICZaA, and other procedures were as described in example 2 to construct pPICZaA-heparanase III recombinant expression vector.
EXAMPLE 4 eukaryotic expression vector was pPICZ A, and other procedures were as described in example 2 to construct a pPICZ A-heparanase III recombinant expression vector
Example 5 expression of heparanase III
Preparation of competent cells by taking the recombinant expression vectors of example 2, example 3 and example 4 and electrotransformation, cell culture medium-induced expression reference Pichia Pink TM The carrier kit instructions were run.
EXAMPLE 6 disruption pretreatment of heparinase III
(a) Centrifuging the thallus fermentation broth induced to be expressed in the embodiment 5 at a low temperature of 4500g and at a temperature of 5 ℃ for 15min;
(b) Collecting supernatant, suspending each 100ml of the bacterial cells obtained in the step (a) by using 1ml Buffer W, and adding a proper amount of protease inhibitor if necessary;
(c) Sonicating cells on an ice-water mixture;
(d) And (c) centrifuging the suspension obtained in the step (c) at 13000rpm at 4 ℃ for 15min.
Preferably, the suspension is viscous, and RNase A (10 ug/ml) and Dnase I (5 ug/ml) may be added and incubated on ice for 10-15min.
EXAMPLE 7 purification of heparanase III
(a) The Srep-Tactin column was washed with Buffer W of 2CVs, and then the suspension obtained in example 6 was subjected to a column loading operation;
(b) Washing the column by using a Buffer W with a concentration of 5CV, and collecting eluent;
(c) Buffer E was added at 6 times 0.5CVs and collected at every 0.5 CV.
(d) The D-sulfate biotin was removed 3 times using Buffer R of 15CVs, and after complete removal, the column was washed with 8CVs Buffer W.
EXAMPLE 8SDS-PAGE detection of heparinase III
The sample treated with Buffer E in example 7 was collected and 20uL was subjected to heparinase III SDS-PAGE to verify that the molecular weight of heparinase III was approximately 73.36kDa as shown in FIG. 1. After analysis by adopting gel density scanning software Bandscan 5.0, the protein purity of target heparanase III corresponding to the amino acid sequence shown by the Seq ID No.2 or the Seq ID No.3 can reach more than 85 percent.
Example 9 enzyme Activity assay
The scanning wavelength was 232nm for 3min. 1000uL of reaction buffer solution (20mM Tris,200mM NaCl,pH =7.4) and enzyme solution (the proportion is regulated according to the activity of the enzyme solution), 500uL of substrate solution (17mM Tris,44mM NaCl,3.5mM CaCl2,25g/L heparin sodium pH=7.4) are placed in a quartz cuvette, uniformly mixed and placed in a spectrophotometer for scanning (the reaction buffer solution and the substrate solution are preheated to 30 ℃ in a water bath), the scanning time is 70s, data in a time period of 40-60s are taken, and the slope k (min) of a curve is calculated -1 ) The enzyme activity (IU/L) of heparinase III is calculated as follows (V is the volume of enzyme solution added into the reaction system):
the enzyme activity of the modified heparanase III (Seq ID No. 3) is 473.68IU/L, and the catalytic activity is not reduced.
Example 10 stability analysis
The purified heparanase III was placed in a constant temperature incubator at 30℃and a certain amount of enzyme solution was regularly aspirated, and the enzyme activity was measured as described in example 9, and compared with that of unmodified heparanase III, and the results are shown in FIG. 2. In FIG. 2, the enzyme sequence 1 represents the protein composed of the original amino acids, the enzyme sequence 2 represents the protein composed of the Seq ID No.2 in the sequence table, and the enzyme sequence 3 represents the protein composed of the Seq ID No.3 in the sequence table.
Compared with the sequence 1 enzyme, the stability of the modified heparanase III is obviously improved, the enzyme activity half-life of the heparanase III corresponding to the sequence 2 enzyme is improved from about 30 hours to about 47 hours, and the stability of the heparanase III corresponding to the mutant sequence 3 enzyme is more obviously improved.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
/>
/>
/>
Sequence listing
<110> Liu Ying
<120> heparanase III and nucleotide sequence encoding the same, recombinant vector and host cell comprising the nucleotide sequence, and use thereof
<130> TPE01497
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 644
<212> PRT
<213> artificial sequence
<220>
<223> manual sequence description: synthetic sequences
<400> 1
Met Gln Ser Ser Ser Ile Thr Arg Lys Asp Phe Asp His Ile Asn Leu
1 5 10 15
Glu Tyr Ser Gly Leu Glu Lys Val Asn Lys Ala Val Ala Ala Gly Asn
20 25 30
Tyr Asp Asp Ala Ala Lys Ala Leu Leu Ala Tyr Tyr Arg Glu Lys Ser
35 40 45
Lys Ala Arg Glu Pro Asp Phe Ser Asn Ala Glu Lys Pro Ala Asp Ile
50 55 60
Arg Gln Pro Ile Asp Lys Val Thr Arg Glu Met Ala Asp Lys Ala Leu
65 70 75 80
Val His Gln Phe Gln Pro His Lys Gly Tyr Gly Tyr Phe Asp Tyr Gly
85 90 95
Lys Asp Ile Asn Trp Gln Met Trp Pro Val Lys Asp Asn Glu Val Arg
100 105 110
Trp Gln Leu His Arg Val Lys Trp Trp Gln Ala Met Ala Leu Val Tyr
115 120 125
His Ala Thr Gly Asp Glu Lys Tyr Ala Arg Glu Trp Val Tyr Gln Tyr
130 135 140
Ser Asp Trp Ala Arg Lys Asn Pro Leu Gly Leu Ser Gln Asp Asn Asp
145 150 155 160
Lys Phe Val Trp Arg Pro Leu Glu Val Ser Asp Arg Val Gln Ser Leu
165 170 175
Pro Pro Thr Phe Ser Leu Phe Val Asn Ser Pro Ala Phe Thr Pro Ala
180 185 190
Phe Leu Met Glu Phe Leu Asn Ser Tyr His Gln Gln Ala Asp Tyr Leu
195 200 205
Ser Thr His Tyr Ala Glu Gln Gly Asn His Arg Leu Phe Glu Ala Gln
210 215 220
Arg Asn Leu Phe Ala Gly Val Ser Phe Pro Glu Phe Lys Asp Ser Pro
225 230 235 240
Arg Trp Arg Gln Thr Gly Ile Ser Val Leu Asn Thr Glu Ile Lys Lys
245 250 255
Gln Val Tyr Ala Asp Gly Met Gln Phe Glu Leu Ser Pro Ile Tyr His
260 265 270
Val Ala Ala Ile Asp Ile Phe Leu Lys Ala Tyr Gly Ser Ala Lys Arg
275 280 285
Val Asn Leu Glu Lys Glu Phe Pro Gln Ser Tyr Val Gln Thr Val Glu
290 295 300
Asn Met Ile Met Ala Leu Ile Ser Ile Ser Leu Pro Asp Tyr Asn Thr
305 310 315 320
Pro Met Phe Gly Asp Ser Trp Ile Thr Asp Lys Asn Phe Arg Met Ala
325 330 335
Gln Phe Ala Ser Trp Ala Arg Val Phe Pro Ala Asn Gln Ala Ile Lys
340 345 350
Tyr Phe Ala Thr Asp Gly Lys Gln Gly Lys Ala Pro Asn Phe Leu Ser
355 360 365
Lys Ala Leu Ser Asn Ala Gly Phe Tyr Thr Phe Arg Ser Gly Trp Asp
370 375 380
Lys Asn Ala Thr Val Met Val Leu Lys Ala Ser Pro Pro Gly Glu Phe
385 390 395 400
His Ala Gln Pro Asp Asn Gly Thr Phe Glu Leu Phe Ile Lys Gly Arg
405 410 415
Asn Phe Thr Pro Asp Ala Gly Val Phe Val Tyr Ser Gly Asp Glu Ala
420 425 430
Ile Met Lys Leu Arg Asn Trp Tyr Arg Gln Thr Arg Ile His Ser Thr
435 440 445
Leu Thr Leu Asp Asn Gln Asn Met Val Ile Thr Lys Ala Arg Gln Asn
450 455 460
Lys Trp Glu Thr Gly Asn Asn Leu Asp Val Leu Thr Tyr Thr Asn Pro
465 470 475 480
Ser Tyr Pro Asn Leu Asp His Gln Arg Ser Val Leu Phe Ile Asn Lys
485 490 495
Lys Tyr Phe Leu Val Ile Asp Arg Ala Ile Gly Glu Ala Thr Gly Asn
500 505 510
Leu Gly Val His Trp Gln Leu Lys Glu Asp Ser Asn Pro Val Phe Asp
515 520 525
Lys Thr Lys Asn Arg Val Tyr Thr Thr Tyr Arg Asp Gly Asn Asn Leu
530 535 540
Met Ile Gln Ser Leu Asn Ala Asp Arg Thr Ser Leu Asn Glu Glu Glu
545 550 555 560
Gly Lys Val Ser Tyr Val Tyr Asn Lys Glu Leu Lys Arg Pro Ala Phe
565 570 575
Val Phe Glu Lys Pro Lys Lys Asn Ala Gly Thr Gln Asn Phe Val Ser
580 585 590
Ile Val Tyr Pro Tyr Asp Gly Gln Lys Ala Pro Glu Ile Ser Ile Arg
595 600 605
Glu Asn Lys Gly Asn Asp Phe Glu Lys Gly Lys Leu Asn Leu Thr Leu
610 615 620
Thr Ile Asn Gly Lys Gln Gln Leu Val Leu Val Pro Trp Ser His Pro
625 630 635 640
Gln Phe Glu Lys
<210> 2
<211> 644
<212> PRT
<213> artificial sequence
<220>
<223> manual sequence description: synthetic sequences
<400> 2
Met Gln Ser Ser Ser Ile Thr Arg Lys Asp Phe Asp His Ile Asn Leu
1 5 10 15
Glu Tyr Ser Gly Leu Glu Lys Val Asn Lys Ala Val Ala Ala Gly Asn
20 25 30
Tyr Asp Asp Ala Ala Lys Ala Leu Leu Ala Tyr Tyr Arg Glu Lys Ser
35 40 45
Lys Ala Arg Glu Pro Asp Phe Ser Asn Ala Glu Lys Pro Ala Asp Ile
50 55 60
Arg Gln Pro Ile Asp Lys Val Thr Arg Glu Met Ala Asp Lys Ala Leu
65 70 75 80
Val His Gln Phe Ala Pro His Lys Gly Tyr Gly Tyr Phe Asp Tyr Gly
85 90 95
Lys Asp Ile Asn Trp Gln Met Trp Pro Val Lys Asp Asn Glu Val Arg
100 105 110
Trp Ala Leu His Arg Val Lys Trp Trp Gln Ala Met Ala Leu Val Tyr
115 120 125
His Ala Thr Gly Asp Glu Lys Tyr Ala Arg Glu Trp Val Tyr Gln Tyr
130 135 140
Ser Asp Trp Ala Arg Lys Asn Pro Leu Gly Leu Ser Gln Asp Asn Asp
145 150 155 160
Lys Phe Val Trp Arg Pro Leu Glu Val Ser Asp Arg Val Gln Ser Leu
165 170 175
Pro Pro Thr Phe Ser Leu Phe Val Asn Ser Pro Ala Phe Thr Pro Ala
180 185 190
Phe Leu Met Glu Phe Leu Asn Ser Tyr His Gln Gln Ala Asp Tyr Leu
195 200 205
Ser Thr His Tyr Ala Glu Gln Gly Asn His Arg Leu Phe Glu Ala Gln
210 215 220
Arg Asn Leu Phe Ala Gly Val Ser Phe Pro Glu Phe Lys Asp Ser Pro
225 230 235 240
Arg Trp Arg Gln Thr Gly Ile Ser Val Leu Asn Thr Glu Ile Lys Lys
245 250 255
Gln Val Tyr Ala Asp Gly Met Gln Phe Glu Leu Ser Pro Ile Tyr His
260 265 270
Val Ala Ala Ile Asp Ile Phe Leu Lys Ala Tyr Gly Ser Ala Lys Arg
275 280 285
Val Asn Leu Glu Lys Glu Phe Pro Gln Ser Tyr Val Gln Thr Val Glu
290 295 300
Asn Met Ile Met Ala Leu Ile Ser Ile Ser Leu Pro Asp Tyr Asn Thr
305 310 315 320
Pro Met Phe Gly Asp Ser Trp Ile Thr Asp Lys Asn Phe Arg Met Ala
325 330 335
Gln Phe Ala Ser Trp Ala Arg Val Phe Pro Ala Asn Gln Ala Ile Lys
340 345 350
Tyr Phe Ala Thr Asp Gly Lys Gln Gly Lys Ala Pro Asn Phe Leu Ser
355 360 365
Lys Ala Leu Ser Asn Ala Gly Phe Tyr Thr Phe Arg Ser Gly Trp Asp
370 375 380
Lys Asn Ala Thr Val Met Val Leu Lys Ala Ser Pro Pro Gly Glu Phe
385 390 395 400
His Ala Ala Pro Asp Asn Gly Thr Phe Glu Leu Phe Ile Lys Gly Arg
405 410 415
Asn Phe Thr Pro Asp Ala Gly Val Phe Val Tyr Ser Gly Asp Glu Ala
420 425 430
Ile Met Lys Leu Arg Asn Trp Tyr Arg Gln Thr Arg Ile His Ser Thr
435 440 445
Leu Thr Leu Asp Asn Gln Asn Met Val Ile Thr Lys Ala Arg Gln Asn
450 455 460
Lys Trp Glu Thr Gly Asn Asn Leu Asp Val Leu Thr Tyr Thr Asn Pro
465 470 475 480
Ser Tyr Pro Asn Leu Asp His Gln Arg Ser Val Leu Phe Ile Asn Lys
485 490 495
Lys Tyr Phe Leu Val Ile Asp Arg Ala Ile Gly Glu Ala Thr Gly Asn
500 505 510
Leu Gly Val His Trp Gln Leu Lys Glu Asp Ser Asn Pro Val Phe Asp
515 520 525
Lys Thr Lys Asn Arg Val Tyr Thr Thr Tyr Arg Asp Gly Asn Asn Leu
530 535 540
Met Ile Ala Ser Leu Asn Ala Asp Arg Thr Ser Leu Asn Glu Glu Glu
545 550 555 560
Gly Lys Val Ser Tyr Val Tyr Asn Lys Glu Leu Lys Arg Pro Ala Phe
565 570 575
Val Phe Glu Lys Pro Lys Lys Asn Ala Gly Thr Gln Asn Phe Val Ser
580 585 590
Ile Val Tyr Pro Tyr Asp Gly Gln Lys Ala Pro Glu Ile Ser Ile Arg
595 600 605
Glu Asn Lys Gly Asn Asp Phe Glu Lys Gly Lys Leu Asn Leu Thr Leu
610 615 620
Thr Ile Asn Gly Lys Gln Gln Leu Val Leu Val Pro Trp Ser His Pro
625 630 635 640
Gln Phe Glu Lys
<210> 3
<211> 644
<212> PRT
<213> artificial sequence
<220>
<223> manual sequence description: synthetic sequences
<400> 3
Met Gln Ser Ser Ser Ile Thr Arg Lys Asp Phe Asp His Ile Asn Leu
1 5 10 15
Glu Tyr Ser Gly Leu Glu Lys Val Asn Lys Ala Val Ala Ala Gly Asn
20 25 30
Tyr Asp Asp Ala Ala Lys Ala Leu Leu Ala Tyr Tyr Arg Glu Lys Ser
35 40 45
Lys Ala Arg Glu Pro Asp Phe Ser Asn Ala Glu Lys Pro Ala Asp Ile
50 55 60
Arg Gln Pro Ile Asp Lys Val Thr Arg Glu Met Ala Asp Lys Ala Leu
65 70 75 80
Val His Gln Phe Ala Pro His Lys Gly Tyr Gly Tyr Phe Asp Tyr Gly
85 90 95
Lys Asp Ile Asn Trp Gln Met Trp Pro Val Lys Asp Asn Glu Val Arg
100 105 110
Trp Ala Leu His Arg Val Lys Trp Trp Gln Ala Met Ala Leu Val Tyr
115 120 125
His Ala Thr Gly Asp Glu Lys Tyr Ala Arg Glu Trp Val Tyr Gln Tyr
130 135 140
Ser Asp Trp Ala Arg Lys Asn Pro Leu Gly Leu Ser Gln Asp Asn Asp
145 150 155 160
Lys Phe Val Trp Arg Pro Leu Glu Val Ser Asp Arg Val Gln Ser Leu
165 170 175
Pro Pro Thr Phe Ser Leu Phe Val Asn Ser Pro Ala Phe Thr Pro Ala
180 185 190
Phe Leu Met Glu Phe Leu Asn Ser Tyr His Gln Gln Ala Asp Tyr Leu
195 200 205
Ser Thr His Tyr Ala Glu Gln Gly Asn His Arg Leu Phe Glu Ala Gln
210 215 220
Arg Asn Leu Phe Ala Gly Val Ser Phe Pro Glu Phe Lys Asp Ser Pro
225 230 235 240
Arg Trp Arg Gln Thr Gly Ile Ser Val Leu Asn Thr Glu Ile Lys Lys
245 250 255
Gln Val Tyr Ala Asp Gly Met Gln Phe Glu Leu Ser Pro Ile Tyr His
260 265 270
Val Ala Ala Ile Asp Ile Phe Leu Lys Ala Tyr Gly Ser Ala Lys Arg
275 280 285
Val Asn Leu Glu Lys Glu Phe Pro Gln Ser Tyr Val Gln Thr Val Glu
290 295 300
Asn Met Ile Met Ala Leu Ile Ser Ile Ser Leu Pro Asp Tyr Asn Thr
305 310 315 320
Pro Met Phe Gly Asp Ser Trp Ile Thr Asp Lys Asn Phe Arg Met Ala
325 330 335
Gln Phe Ala Ser Trp Ala Arg Val Phe Pro Ala Asn Gln Ala Ile Lys
340 345 350
Tyr Phe Ala Thr Asp Gly Lys Gln Gly Lys Ala Pro Asn Phe Leu Ser
355 360 365
Lys Ala Leu Ser Asn Ala Gly Phe Tyr Thr Phe Arg Ser Gly Trp Asp
370 375 380
Lys Asn Ala Thr Val Met Val Leu Lys Ala Ser Pro Pro Gly Glu Phe
385 390 395 400
His Ala Val Pro Asp Asn Gly Thr Phe Glu Leu Phe Ile Lys Gly Arg
405 410 415
Asn Phe Thr Pro Asp Ala Gly Val Phe Val Tyr Ser Gly Asp Glu Ala
420 425 430
Ile Met Lys Leu Arg Asn Trp Tyr Arg Gln Thr Arg Ile His Ser Thr
435 440 445
Leu Thr Leu Asp Asn Gln Asn Met Val Ile Thr Lys Ala Arg Gln Asn
450 455 460
Lys Trp Glu Thr Gly Asn Asn Leu Asp Val Leu Thr Tyr Thr Asn Pro
465 470 475 480
Ser Tyr Pro Asn Leu Asp His Gln Arg Ser Val Leu Phe Ile Asn Lys
485 490 495
Lys Tyr Phe Leu Val Ile Asp Arg Ala Ile Gly Glu Ala Thr Gly Asn
500 505 510
Leu Gly Val His Trp Gln Leu Lys Glu Asp Ser Asn Pro Val Phe Asp
515 520 525
Lys Thr Lys Asn Arg Val Tyr Thr Thr Tyr Arg Asp Gly Asn Asn Leu
530 535 540
Met Ile Ala Ser Leu Asn Ala Asp Arg Thr Ser Leu Asn Glu Glu Glu
545 550 555 560
Gly Lys Val Ser Tyr Val Tyr Asn Lys Glu Leu Lys Arg Pro Ala Phe
565 570 575
Val Phe Glu Lys Pro Lys Lys Asn Ala Gly Thr Gln Asn Phe Val Ser
580 585 590
Ile Val Tyr Pro Tyr Asp Gly Gln Lys Ala Pro Glu Ile Ser Ile Arg
595 600 605
Glu Asn Lys Gly Asn Asp Phe Glu Lys Gly Lys Leu Asn Leu Thr Leu
610 615 620
Thr Ile Asn Gly Lys Gln Gln Leu Val Leu Val Pro Trp Ser His Pro
625 630 635 640
Gln Phe Glu Lys
<210> 4
<211> 1935
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic sequences
<400> 4
atgcaatctt cttctatcac tagaaaggat ttcgatcata tcaacttgga gtattctggt 60
ttggaaaagg ttaacaaagc tgttgctgct ggtaattacg atgatgctgc taaggctttg 120
ttggcttact atagagagaa gtctaaagct agagaaccag atttttctaa tgctgagaag 180
ccagctgata tcagacaacc tatcgataag gttactagag aaatggctga taaggctttg 240
gttcatcaat tcgctccaca caagggttac ggttacttcg attacggtaa agatatcaac 300
tggcaaatgt ggcctgttaa ggataacgaa gttagatggg ctttgcatcg tgttaagtgg 360
tggcaagcta tggctttggt ttatcatgct actggagatg agaagtatgc tagagaatgg 420
gtttaccaat attctgattg ggctagaaag aacccattgg gtttgtctca agataacgat 480
aagttcgttt ggagaccttt ggaggtttct gatagagttc aatctttgcc acctactttt 540
tctttgttcg ttaactctcc agcttttact cctgctttct tgatggaatt tttgaactct 600
taccatcaac aagctgatta cttgtctact cattatgctg agcaaggtaa ccacagattg 660
ttcgaagctc aaagaaattt gtttgctggt gtttcttttc cagagtttaa ggattctcct 720
agatggagac aaactggtat ctctgttttg aacactgaga ttaagaaaca agtttacgct 780
gatggtatgc aattcgaatt gtctccaatc taccacgttg ctgctattga tattttcttg 840
aaggcttacg gttctgctaa aagagttaac ttggagaagg aatttcctca atcttacgtt 900
caaactgttg aaaacatgat catggctttg atctctattt ctttgccaga ttacaacact 960
cctatgtttg gagattcttg gatcactgat aagaacttca gaatggctca atttgcttct 1020
tgggctagag ttttcccagc taaccaagct attaaatact tcgctactga tggtaaacag 1080
ggtaaagctc ctaacttctt gtctaaggct ttgtctaatg ctggttttta cactttcaga 1140
tctggttggg ataaaaatgc tactgttatg gttttgaagg cttctccacc tggagagttt 1200
catgctgctc cagataacgg tactttcgaa ttgttcatta agggtagaaa cttcactcct 1260
gatgctggtg tttttgttta ttctggagat gaggctatta tgaagttgag aaactggtac 1320
agacaaacta gaatccattc tactttgact ttggataacc aaaacatggt tattactaag 1380
gctagacaaa acaagtggga aactggtaac aatttggatg ttttgactta cactaaccca 1440
tcttatccta atttggatca tcaaagatct gttttgttca ttaacaagaa atactttttg 1500
gttattgata gagctatcgg agaggctact ggtaatttgg gtgttcactg gcaattgaag 1560
gaagattcta acccagtttt cgataagact aaaaatagag tttacactac ttacagagat 1620
ggtaacaatt tgatgattgc ttctttgaac gctgatagaa cttctttgaa tgaagaggag 1680
ggtaaagttt cttacgttta caataaggag ttgaaaagac cagcttttgt tttcgaaaag 1740
cctaagaaaa acgctggtac tcaaaacttc gtttctatcg tttacccata tgatggtcaa 1800
aaagctcctg agatctctat cagagaaaac aagggtaacg atttcgagaa gggtaaattg 1860
aacttgactt tgactattaa tggtaaacaa caattggttt tggttccatg gtctcaccct 1920
caatttgaaa agtaa 1935
<210> 5
<211> 1935
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic sequences
<400> 5
atgcaatctt cttctatcac tagaaaggat ttcgatcata tcaacttgga gtattctggt 60
ttggaaaagg ttaacaaagc tgttgctgct ggtaattacg atgatgctgc taaggctttg 120
ttggcttact atagagagaa gtctaaagct agagaaccag atttttctaa tgctgagaag 180
ccagctgata tcagacaacc tatcgataag gttactagag aaatggctga taaggctttg 240
gttcatcaat tcgctccaca caagggttac ggttacttcg attacggtaa agatatcaac 300
tggcaaatgt ggcctgttaa ggataacgaa gttagatggg ctttgcatcg tgttaagtgg 360
tggcaagcta tggctttggt ttatcatgct actggagatg agaagtatgc tagagaatgg 420
gtttaccaat attctgattg ggctagaaag aacccattgg gtttgtctca agataacgat 480
aagttcgttt ggagaccttt ggaggtttct gatagagttc aatctttgcc acctactttt 540
tctttgttcg ttaactctcc agcttttact cctgctttct tgatggaatt tttgaactct 600
taccatcaac aagctgatta cttgtctact cattatgctg agcaaggtaa ccacagattg 660
ttcgaagctc aaagaaattt gtttgctggt gtttcttttc cagagtttaa ggattctcct 720
agatggagac aaactggtat ctctgttttg aacactgaga ttaagaaaca agtttacgct 780
gatggtatgc aattcgaatt gtctccaatc taccacgttg ctgctattga tattttcttg 840
aaggcttacg gttctgctaa aagagttaac ttggagaagg aatttcctca atcttacgtt 900
caaactgttg aaaacatgat catggctttg atctctattt ctttgccaga ttacaacact 960
cctatgtttg gagattcttg gatcactgat aagaacttca gaatggctca atttgcttct 1020
tgggctagag ttttcccagc taaccaagct attaaatact tcgctactga tggtaaacag 1080
ggtaaagctc ctaacttctt gtctaaggct ttgtctaatg ctggttttta cactttcaga 1140
tctggttggg ataaaaatgc tactgttatg gttttgaagg cttctccacc tggagagttt 1200
catgctgttc cagataacgg tactttcgaa ttgttcatta agggtagaaa cttcactcct 1260
gatgctggtg tttttgttta ttctggagat gaggctatta tgaagttgag aaactggtac 1320
agacaaacta gaatccattc tactttgact ttggataacc aaaacatggt tattactaag 1380
gctagacaaa acaagtggga aactggtaac aatttggatg ttttgactta cactaaccca 1440
tcttatccta atttggatca tcaaagatct gttttgttca ttaacaagaa atactttttg 1500
gttattgata gagctatcgg agaggctact ggtaatttgg gtgttcactg gcaattgaag 1560
gaagattcta acccagtttt cgataagact aaaaatagag tttacactac ttacagagat 1620
ggtaacaatt tgatgattgc ttctttgaac gctgatagaa cttctttgaa tgaagaggag 1680
ggtaaagttt cttacgttta caataaggag ttgaaaagac cagcttttgt tttcgaaaag 1740
cctaagaaaa acgctggtac tcaaaacttc gtttctatcg tttacccata tgatggtcaa 1800
aaagctcctg agatctctat cagagaaaac aagggtaacg atttcgagaa gggtaaattg 1860
aacttgactt tgactattaa tggtaaacaa caattggttt tggttccatg gtctcaccct 1920
caatttgaaa agtaa 1935
Claims (12)
1. A heparanase III, comprising an amino acid sequence as shown in Seq ID No.2 or Seq ID No. 3.
2. A nucleotide sequence encoding heparanase III according to claim 1.
3. The nucleotide sequence according to claim 2, characterized in that the nucleotide sequence is shown as Seq ID No.4 or Seq ID No. 5.
4. A recombinant vector comprising the nucleotide sequence of claim 2 or 3.
5. The recombinant vector according to claim 4, wherein the recombinant vector is a eukaryotic recombinant vector.
6. The recombinant vector according to claim 5, wherein the eukaryotic cell recombinant vector is any one of pPink-HC, pPICZaA, and pPICZ A.
7. The recombinant vector of claim 6, wherein the eukaryotic recombinant vector is pPink-HC.
8. A host cell comprising the recombinant vector of any one of claims 4 to 7.
9. The host cell of claim 8, wherein the host cell is pichia pastoris.
10. The host cell of claim 9, wherein the host cell is a cell,
the pichia pastoris is X33.
11. A process for the preparation of heparanase III according to claim 1, comprising the steps of:
(a) Synthesizing a nucleotide sequence encoding heparanase III of claim 1;
(b) Combining the nucleotide sequence in (a) with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;
(c) Introducing the recombinant vector in (b) into a host cell, culturing and inducing expression, and purifying to obtain the heparanase III.
12. A process according to claim 11, wherein step (c) uses Buffer W to purify the Srep-Tactin column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210203224.7A CN114703168B (en) | 2022-03-02 | 2022-03-02 | Heparinase III |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210203224.7A CN114703168B (en) | 2022-03-02 | 2022-03-02 | Heparinase III |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114703168A CN114703168A (en) | 2022-07-05 |
| CN114703168B true CN114703168B (en) | 2023-08-18 |
Family
ID=82167546
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210203224.7A Active CN114703168B (en) | 2022-03-02 | 2022-03-02 | Heparinase III |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114703168B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995034635A1 (en) * | 1994-06-10 | 1995-12-21 | Ibex Technologies | Nucleic acid sequences and expression systems for heparinase ii and heparinase iii derived from flavobacterium heparinum |
| WO2001066772A2 (en) * | 2000-03-08 | 2001-09-13 | Massachusetts Institute Of Technology | Heparinase iii and uses thereof |
| CN101942025A (en) * | 2010-08-20 | 2011-01-12 | 清华大学 | Heparanase III fusion protein and coding gene and expression method thereof |
| CN110343691A (en) * | 2019-07-24 | 2019-10-18 | 宝锐生物科技泰州有限公司 | Saltant type heparinase I and its coding nucleotide sequence, the recombinant vector including the nucleotide sequence and host cell and application |
| CN113025598A (en) * | 2021-01-25 | 2021-06-25 | 上海宝维医药技术有限公司 | Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO _ heparinase III fusion protein prepared by method |
-
2022
- 2022-03-02 CN CN202210203224.7A patent/CN114703168B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995034635A1 (en) * | 1994-06-10 | 1995-12-21 | Ibex Technologies | Nucleic acid sequences and expression systems for heparinase ii and heparinase iii derived from flavobacterium heparinum |
| WO2001066772A2 (en) * | 2000-03-08 | 2001-09-13 | Massachusetts Institute Of Technology | Heparinase iii and uses thereof |
| CN101942025A (en) * | 2010-08-20 | 2011-01-12 | 清华大学 | Heparanase III fusion protein and coding gene and expression method thereof |
| CN110343691A (en) * | 2019-07-24 | 2019-10-18 | 宝锐生物科技泰州有限公司 | Saltant type heparinase I and its coding nucleotide sequence, the recombinant vector including the nucleotide sequence and host cell and application |
| CN113025598A (en) * | 2021-01-25 | 2021-06-25 | 上海宝维医药技术有限公司 | Method for preparing recombinant heparinase III by utilizing SUMO fusion expression system and SUMO _ heparinase III fusion protein prepared by method |
Non-Patent Citations (1)
| Title |
|---|
| Hashimoto,W. 等.ACCESSION：4MMH_A，"Chain A, Heparinase III protein".《Genbank》.2020,feature、origin部分. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114703168A (en) | 2022-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Beard et al. | [11] Purification and domain-mapping of mammalian DNA polymerase β | |
| CN108239633B (en) | Mutant of D-psicose-3-epimerase with improved catalytic activity and application thereof | |
| CN111718949B (en) | Introduction of unnatural amino acids in proteins using a two-plasmid system | |
| CN111850020B (en) | Introduction of unnatural amino acids in proteins using plasmid systems | |
| AU2020242724B2 (en) | Aminoacyl-tRNA synthetase for efficiently introducing lysine derivative in protein | |
| CN109055339B (en) | TEV protease mutant, gene, biological material, preparation method, reagent or kit and application | |
| CN109136209B (en) | Enterokinase light chain mutant and application thereof | |
| JP2023506154A (en) | Allulose epimerization enzyme mutant, method for producing the same, and method for producing allulose using the same | |
| CN114703168B (en) | Heparinase III | |
| CN113234704B (en) | Method for preparing recombinant serratia marcescens nuclease | |
| CN116478969A (en) | Collagenase mutant, method for promoting secretory expression of recombinant collagenase and application thereof | |
| US7109015B2 (en) | Removal of N-terminal methionine from proteins by engineered methionine aminopeptidase | |
| CN113061598A (en) | Trypsin mutant, preparation method and application thereof | |
| JP2517861B2 (en) | Method for producing immobilized enzyme | |
| CN113913413B (en) | Salt-tolerant RPK mutant and application thereof | |
| CN113652409B (en) | Novel glycyrrhetinic acid glucuronyl transferase mutant and application thereof | |
| CN114990097B (en) | L-aspartic acid-alpha-decarboxylase mutant and application thereof | |
| KR20220162226A (en) | A novel fusion tag system promising soluble expression and purification in Escherichia coli using CBM66 and levan, and their applications | |
| CN117586415A (en) | Recombinant Ulp1 fusion protease and preparation method thereof | |
| CN113913414A (en) | Double-base enzyme Kex2 mutant with high stability and high catalytic efficiency | |
| CN117025557A (en) | Cyclooxygenase Lsd18 variant with higher heat stability and stronger enzyme activity and application thereof | |
| KR101646728B1 (en) | A method of synthesizing unnatural protein using degenercy reprogramming | |
| CN115873837A (en) | High-expression novel phenylalanine ammonia lyase | |
| CN117587041A (en) | Recombinant uricase, and preparation method and application thereof | |
| CN118006578A (en) | Chondroitin 4-O-sulfuric acid transferase with improved enzyme activity and production method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231005 Address after: 101204 No. 739-1927, zone 1, Mafang Industrial Park, Zhongguancun Science Park, Pinggu District, Beijing Patentee after: Beijing ed Hauck International Technology Co.,Ltd. Address before: 101399 room 505, block D, Nanfaxin Xuhui airport, Shunyi District, Beijing Patentee before: Liu Ying |
|
| TR01 | Transfer of patent right |