CN103930544A

CN103930544A - Methods for production of archeae protease in yeast

Info

Publication number: CN103930544A
Application number: CN201280054844.3A
Authority: CN
Inventors: 松井知子
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2011-11-08
Filing date: 2012-11-05
Publication date: 2014-07-16
Also published as: WO2013068309A1; EP2794873A1

Abstract

The invention relates to: a method for producing a protease in yeast, comprising providing a yeast host cell comprising at least one polynucleotide expression construct encoding a first polypeptide secreted by the host cell in translational fusion with a second polypeptide, wherein the second polypeptide is a protease having a mature amino acid sequence similar to that of SEQ ID NO:2; the corresponding yeast host cell and the expression construct.

Description

In yeast, produce the method for archeobacteria proteolytic enzyme

to quoting of sequence table

The sequence table that the application comprises computer-reader form.This computer-reader form is incorporated to herein with for referencial use.

Technical field

The present invention relates to by expressed fusion protein microorganisms producing protease polypeptide in yeast.The production exemplifying is below for being derived from the proteolytic enzyme of the fierce fireball bacterium of archeobacteria (Pyrococcus furiosus).

Background technology

Suitability for industrialized production albumen or polypeptide in many ways at present, all these all relate to the cultivation of raw albuminiferous microorganism.The several methods of optimizing industrial protein yield be in the art know and just utilized routinely, such as handle growth medium, transformation microorganism such as by impact, expect albumen encoding gene expression to genetic elements as sudden change, the variation of promotor and signal sequence etc., and operator gene itself, so that for example Enhancin stability or activity.

Previously describe fusion rotein and as other modes of production, be difficult to a kind of mode of the albumen of acquisition, an example can be active people's antibody (Goshorn, S.C. wait people, Cancer Research, 1993, Vol.53 (9) pp.2123-2127) manufacture enzyme (WO00/75344) or in bacillus host.

Summary of the invention

Enzyme from extreme microorganism usually has the interested character of industrial application, for example, and very high thermostability.Yet they may be difficult to the relevant amount of industry and produce with activity form.

The inventor finds, the proteolytic enzyme that derives from the fierce fireball bacterium of archeobacteria is difficult to express in bread yeast (baker ' s yeast), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), even from be designed for yeast and secretion signal (the well running in yeast conventionally of this secretion signal) together be also (referring to embodiment 1) like this while expressing in the synthetic gene of expression.

Yet, be surprisingly found out that, among the carrier proteins of the archeobacteria proteolytic enzyme allos fungus secretion different from three, any one translation is merged and while expressing, proteolytic enzyme can be with it ripe and activity form by yeast saccharomyces cerevisiae, produced and secrete (referring to embodiment 2).

Therefore, in first aspect, the present invention relates to produce the method for proteolytic enzyme in yeast, comprising:

(a) provide the yeast host cell that comprises at least one polynucleotide expression construct, this polynucleotide expression construct coding translates with the second polypeptide the first polypeptide secreted by this host cell merging, and wherein this second polypeptide is selected from:

(i) mature polypeptide of its maturing part and SEQ ID NO:2; Preferably have at least 60% with the position [1-412] (comprising two ends) of SEQ ID NO:2, for example the proteolytic enzyme of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity;

(ii) its maturing part is by the mature polypeptide encoded sequence with SEQ ID NO:1; Preferably have at least 60% with the position [397-1632] (comprising two ends) of SEQ ID NO:1, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or the coded proteolytic enzyme of polynucleotide of 100%DNA sequence identity;

(iii) its maturing part by low wait stringent condition, medium stringent condition, under high stringent condition, high stringent condition or the stringent condition such as high with the mature polypeptide encoded sequence of SEQ ID NO:1; Preferably with the position [397-1632] (comprising two ends) of SEQ ID NO:1; Or the coded proteolytic enzyme of polynucleotide of the total length complement hybridization of any one in both;

(iv) variant of the position [1-412] (comprising two ends) of the mature polypeptide of SEQ ID NO:2, preferred SEQ ID NO:2, it is included in one or more locational replacements, disappearance and/or insertion; With

(v) (i), (ii), (iii) or (iv) fragment with protease activity of polypeptide,

(b) under the condition of expression and secretion that is of value to this first polypeptide, cultivate host cell, secretion is translated fusion polypeptide and is produced ripe proteolytic enzyme thus; With

(c) optionally, reclaim proteolytic enzyme.

In second aspect, the present invention relates to the yeast host cell defined in the step (a) as first aspect.

An aspect in the end, the present invention relates to the polynucleotide expression construct defined in the step (a) as first aspect.

Summary of the invention

production method

A first aspect of the present invention relates to the method for producing proteolytic enzyme in yeast, comprising:

(v) (i), (ii), (iii) or (iv) fragment with protease activity of polypeptide,

(c) optionally, reclaim proteolytic enzyme.

Use methods known in the art to cultivate host cell in being suitable for producing the nutrient media of polypeptide.For example, can be by suitable medium and allowing to carry out shake-flask culture under expression of polypeptides and/or separated condition or carrying out small-scale or large scale fermentation (comprise continuously ferment, batch fermentation, fed-batch fermentation or solid state fermentation) carrys out culturing cell in laboratory or industrial fermentation tank.Use step known in the art to cultivate in comprising the suitable nutrient media of Carbon and nitrogen sources and inorganic salt.Suitable medium can obtain or can prepare according to disclosed composition (for example,, in the catalogue of American type culture collection) from commercial supplier.If polypeptide is secreted in nutrient media, can directly from medium, reclaim polypeptide.If polypeptide is secreted going out not, can from cell lysate, reclaim.

Can detect protease polypeptide by the specific method for protease polypeptide known in field.These detection methods are including, but not limited to use, the formation of enzyme product or the disappearance of enzyme substrates of specific antibody.For example, the mensuration of enzyme can be used to judge the activity of polypeptide.

Useful methods known in the art reclaim polypeptide.For example, can from nutrient media, reclaim polypeptide by conventional step, this step including, but not limited to collecting, centrifugal, filter, extraction, spraying are dry, evaporation or precipitation.

Can obtain substantially pure polypeptide by several different methods purification polypeptide known in the art, the method including, but not limited to chromatography (for example, ion-exchange, affinity, hydrophobicity, chromatofocusing and size exclusion), electrophoretic method (for example, preparation formula isoelectrofocusing), difference dissolution method (for example, ammonium sulfate precipitation), SDS-PAGE or extraction are (referring to for example, Protein Purification, Janson and Ryden, editor, VCH Publishers, New York, 1989).

Aspect alternative, do not reclaim polypeptide, but use the host cell of the present invention of express polypeptide as the source of polypeptide.

variant

A first aspect of the present invention also relates to the variant of the mature polypeptide of the SEQ ID NO:2 that for example, comprises replacement, disappearance in one or more (, several) position and/or insert.In embodiment, the quantity of amino acid substitution, disappearance and/or insertion in the mature polypeptide of introducing SEQ ID NO:2 is no more than 10, as 1,2,3,4,5,6,7,8 or 9.It can be that character is slight that amino acid changes, i.e. not remarkably influenced protein folding and/or active conservative amino acid substitution or insertion; Little disappearance, is generally 1～30 amino acid; Little aminoterminal or carboxyl terminal extend, as N-terminal methionine residues; The little joint peptide of 20～25 residues of as many as; Or by changing net charge or other function, promote the little extension of purifying, as polyhistidine sequence (poly-histidine tract), epitope or binding domains.

The conservative example of replacing is in the group of basic aminoacids (arginine, Methionin and Histidine), acidic amino acid (L-glutamic acid and aspartic acid), polare Aminosaeren (glutamine and l-asparagine), hydrophobic amino acid (leucine, Isoleucine and α-amino-isovaleric acid), aromatic amino acid (phenylalanine, tryptophane and tyrosine) and little amino acid (glycine, L-Ala, Serine, Threonine and methionine(Met)).Conventionally the amino acid substitution that does not change specific activity is known in field, and at for example H.Neurath and R.L.Hill, 1979, In, The Proteins, Academic Press, records in New York.Common replaces with, Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

Or the physico-chemical property that the character that amino acid changes is polypeptide is changed.For example, amino acid change can improve polypeptide thermostability, change substrate specificity, change optimal pH etc.

Can identify the primary amino acid in polypeptide according to step known in field, for example site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science244:1081-1085).In technology described later, in each residue of molecule, introduce single alanine mutation, and active to the mutating molecule test of gained, take and identify that for molecular activity be critical amino-acid residue.Also referring to, the people such as Hilton, 1996, J.Biol.Chem.271:4699-4708.Enzyme or the interactional reactive site of other biology also can be determined by the physical analysis to structure, as passed through following these technology: as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with being measured inferring the contact amino acid whose sudden change in site.Referring to such as people such as deVos, 1992, Science255:306-312; The people such as Smith, 1992, J.Mol.Biol.224:899-904; The people such as Wlodaver, 1992, FEBS Lett.309:59-64.Also can be from comparing to infer the identity (identity) of primary amino acid to relevant polypeptide.

Can use known sudden change, restructuring and/or Shuffling Method, the screening step of being then correlated with (Reidhaar-Olson and Sauer for example, 1988, Science241:53-57; Bowie and Sauer, 1989, Proc.Natl.Acad.Sci.USA86:2152-2156; WO95/17413; Or disclosed in WO95/22625) make and test single or multiple amino acid substitutions, disappearance and/or insertion.Other available method comprises fallibility PCR, phage display (for example, the people such as Lowman, 1991, Biochemistry30:10832-10837; United States Patent (USP) the 5th, 223, No. 409; WO92/06204) and site-directed mutagenesis (people such as Derbyshire, 1986, Gene46:145; The people such as Ner, 1988, DNA7:127).

Sudden change/Shuffling Method can be combined with high-throughout automatically screening method to detect the activity (people such as Ness, 1999, Nature Biotechnology17:893-896) by the clone's of host cell expression mutant polypeptide.The mutant DNA molecule of coding active polypeptide is reclaimed in standard method in can use field from host cell, and order-checking fast.These methods make to determine fast the importance of single amino acids residue in polypeptide.

translation is merged

We mention in background technology part, it is reported and in genus bacillus, successfully produced the enzyme beyond expression of words merging with the native protein pectate lyase translation of height expression and secretion, this pectate lyase is fused to enzyme at its C end place to allow native protein as carrier or traction apparatus (tractor) (WO00/75344).

Similarly, protease polypeptide of the present invention is expressed as to fusion polypeptide, wherein the polypeptide of secretion merges the N end to proteolytic enzyme of the present invention.

By the polynucleotide of another polypeptide of coding being fused to the polynucleotide of code book invention proteolytic enzyme, prepare fusion polypeptide.The technology of preparation fusion polypeptide is known in the art, and comprise that the encoding sequence that connects coded polypeptide makes them in reading frame and the expression of fusion polypeptide under the control of same promotor and terminator.

It may be favourable between fusion polypeptide, comprising little peptide linker (linker), so as physically to allow them correctly to fold and make this fusion polypeptide its secretion after may be even easily spontaneously to cut, to discharge proteolytic enzyme of the present invention.

If fusion polypeptide is unripe to discharge ripe proteolytic enzyme voluntarily after secretion, it can further design between two polypeptide to merge in translation and comprise specificity solution Protein cleavage site so.Once secretion fusion rotein, this site is cut to discharge two polypeptide subsequently.The example of cleavage site includes but not limited to, people such as Martin, and 2003, J.Ind.Microbiol.Biotechnol.3:568-576; The people such as Svetina, 2000, J.Biotechnol.76:245-251; The people such as Rasmussen-Wilson, 1997, Appl.Environ.Microbiol.63:3488-3493; The people such as Ward, 1995, Biotechnology13:498-503; And the people such as Contreras, 1991, Biotechnology9:378-381; The people such as Eaton, 1986, Biochemistry25:505-512; The people such as Collins-Racie, 1995, Biotechnology13:982-987; The people such as Carter, 1989, Proteins:Structure, Function, and Genetics6:240-248; And Stevens, disclosed site in 2003, Drug Discovery World4:35-48.

In a preferred embodiment, this at least one polynucleotide expression construct further comprises that coding and the first polypeptide and the translation of the second polypeptide merge and the 3rd polynucleotide of the connection peptides between them; Preferably, the 3rd polynucleotide encoding connection peptides; Preferably, this connection peptides comprises 2～200 amino acid; Preferably 3～100 amino acid, for example 4～50 or 5～25 amino acid; Most preferably, this connection peptides comprises the aminoacid sequence with SEQ ID NO:11 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

In a preferred embodiment, the first polypeptide that translation is merged is the enzyme of natural or allos total length or the brachymemma of C end, be preferably lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, aminopeptidase for example, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase (mutanase), oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase, or xylobiase.

In a preferred embodiment, as illustrated below, the first polypeptide that translation is merged is the glucoamylase of natural or allos total length or the brachymemma of C end; Be preferably the glucose starch enzyme polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

(a) have at least 60% with the mature polypeptide of SEQ ID NO:6, for example the polypeptide of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity;

(b) by low wait stringent condition, medium stringent condition, under high stringent condition, high stringent condition or the stringent condition such as high with (i) the mature polypeptide cDNA encoding sequence of SEQ ID NO:5, (ii) on November 23rd, 2009, be preserved in Germany microorganism and cell culture preservation center (DSMZ) and distribute the AMG gene inserting in the plasmid AMG1 in the coli strain that accession number is DSM23123, or (iii) the coded polypeptide of polynucleotide of (i) or the hybridization of total length complement (ii); With

(c) by the mature polypeptide encoded sequence with SEQ ID NO:5 or with on November 23rd, 2009, be preserved in Germany microorganism and cell culture preservation center (DSMZ) and distribute the mature polypeptide encoded sequence of the AMG gene inserting in the plasmid AMG1 in the coli strain that accession number is DSM23123 to have at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the polypeptide of the polynucleotide encoding of 100% sequence identity.

In another preferred implementation, as illustrated below, the first polypeptide that this translation is merged is the phytase of natural or allos total length or the brachymemma of C end; Be preferably the phytase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

(a) have at least 60% with the mature polypeptide of SEQ ID NO:8, for example the polypeptide of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity;

(b) by low wait stringent condition, medium stringent condition, in the polypeptide coded with (i) the mature polypeptide cDNA encoding sequence of SEQ ID NO:7 or the polynucleotide of its total length complement hybridization under high stringent condition, high stringent condition or the stringent condition such as high; With

(c) by having at least 60% with the mature polypeptide encoded sequence of SEQ ID NO:7, the coded polypeptide of the polynucleotide of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for example.

In another preferred implementation, as illustrated below, the first polypeptide that this translation is merged is the α-amylase of natural or allos total length or the brachymemma of C end; Be preferably the α-amylase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

(a) have at least 60% with the mature polypeptide of SEQ ID NO:10, for example the polypeptide of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity;

(b) by low wait stringent condition, medium stringent condition, in the polypeptide coded with (i) the mature polypeptide cDNA encoding sequence of SEQ ID NO:9 or the polynucleotide of its total length complement hybridization under high stringent condition, high stringent condition or the stringent condition such as high; With

(c) by having at least 60% with the mature polypeptide encoded sequence of SEQ ID NO:9, the coded polypeptide of the polynucleotide of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for example.

the source with the polypeptide of protease activity

The polypeptide with protease activity of the present invention can obtain from the microorganism of any genus.For purposes of the present invention, herein with the term of the associated use in given source " available from " should refer to, by the polypeptide of polynucleotide encoding, be by this source or be wherein inserted with the strain generation of the polynucleotide in this source.On the one hand, available from the polypeptide in given source be secreted into extracellular.

Protease polypeptide can be archeobacteria polypeptide.For example, this polypeptide can be hot-bulb bacterium (Thermococci) guiding principle, such as from hot-bulb bacterium (Thermococcales) order, from hot-bulb bacterium (Thermococcaceae) section or even belong to from fireball bacterium (Pyrococcus).In one aspect, protease polypeptide can be fierce fireball bacterium polypeptide.

Should be understood that, with regard to previously described species, complete and incomplete state has been contained in the present invention, and the equivalent on other taxonomy, and for example what anamorph, is regardless of their known kind names.Those skilled in the art can identify the identity of suitable equivalent at an easy rate.

The public can easily obtain in some preservations mechanism the strain of these species, such mechanism such as American type culture collection (American Type Culture Collection, ATCC), Germany microorganism and cell culture preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Holland (the Centraalbureau Voor Schimmelcultures of DSMZ, CBS) and research centre, North, agricultural research institute's patent culture collection center (Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, NRRL).

Can use above-mentioned probe to originate from other, for example comprise, from the separated microorganism of nature (, soil, compost, water etc.) or the DNA sample evaluation for example, directly obtaining from natural materials (, soil, compost, water etc.) and obtain these polypeptide.Be used for is directly well known in the art from the technology of Natural habitat (habitat) separate microorganism and DNA.Can obtain by similarly screening the genomic dna of another microorganism or the DNA sample of cDNA library or mixing the polynucleotide of coded polypeptide subsequently.By probe in detecting to after the polynucleotide of coded polypeptide, just can by use technical point that those of ordinary skills know from or clone polynucleotide (referring to, for example, the people such as Sambrook, 1989, the same).

polynucleotide

The invention still further relates to separated polynucleotide, its coding is protease polypeptide of the present invention as described herein.

For separating of or the technology of clone's polynucleotide be known in the art, and comprise from genomic dna or cDNA separated, or their combination.Can be by for example using the polymerase chain reaction (PCR) know or the antibody screening of expression library to detect the cloned DNA fragment with apokoinou construction feature, thus realize from genomic dna cloning polynucleotide.Referring to, for example, the people such as Innis, 1990, PCR:A Guide to Methods and Application, Academic Press, New York.Can use other nucleic acid amplification method such as ligase chain reaction (LCR) (LCR), connect to activate and transcribe (ligation activated transcription) amplification (NASBA) (LAT) with based on polynucleotide.Can clone polynucleotide from Pyrococcus bacterial strain or relevant organism, and can be therefore allelic variant or the kind variant (species variant) of for example polypeptid coding area of these polynucleotide.

The polynucleotide of modifying code book invention polypeptide for synthetic and described polypeptide substantially similar polypeptide may be essential.Term refers to polypeptide " substantially similar " form that the non-natural of polypeptide exists.These polypeptide can be different from the mode of some through engineering approaches from its natural origin isolated polypeptide, for example, and the different variants in aspect such as specific activity, thermostability, optimal pH.The polynucleotide that mature polypeptide encoded sequence that can be in the position 397～1632 as SEQ ID NO:1 exists are for example on the basis of its subsequence, and/or replace to build variant by introducing following Nucleotide: described replacement does not cause the change of polypeptid acid sequence, but meet, be intended to for producing the organic codon usage of host of enzyme; Or by introducing following Nucleotide, replace to build variant: described replacement can produce different aminoacid sequences.The general introduction replacing about Nucleotide, referring to, for example, the people such as Ford, 1991, Protein Expression and Purification2:95-107.

nucleic acid construct

A third aspect of the present invention relates to defined polynucleotide expression construct in the step (a) as first aspect.

The invention still further relates to the nucleic acid construct that comprises the polynucleotide of the present invention that are operably connected with one or more control sequences, above-mentioned control sequence guides the expression of encoding sequence in suitable host cell under the condition compatible with this control sequence.

Can handle in several ways polynucleotide, so that the expression of polypeptide to be provided.Depend on expression vector, it can be desirable or essential before polynucleotide are inserted into carrier, it being handled.The technology of using recombinant DNA method to modify polynucleotide is well known in the art.

Control sequence can be promotor, and it is the polynucleotide for the expression of the polynucleotide of code book invention polypeptide by host cell identification.Promotor contains the transcriptional control sequence that mediates expression of polypeptides.Promotor can be any polynucleotide that show transcriptional activity in host cell, the promotor that comprises natural or allos and heterozygosis sudden change, brachymemma, and can from the Codocyte with host cell homology or allos or in cell, obtain by the gene of polypeptide.

In yeast host, available promotor is available from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae galactokinase (GAL1), yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), yeast saccharomyces cerevisiae triosephosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and the kinase whose gene of yeast saccharomyces cerevisiae 3-phoshoglyceric acid.Other promotor that can be used for yeast host cell is by people such as Romanos, and 1992, Yeast8:423-488 describes.

Control sequence can be also transcription terminator, and it is identified to stop transcribing by host cell.Terminator is operably connected with 3 ' end of the polynucleotide of coded polypeptide.Any terminator working in host cell all can be used for the present invention.

Be used for the preferred terminator of yeast host cell available from the gene of yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate, brewing yeast cell pigment C (CYC1) and yeast saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.Other terminators that can be used for yeast host cell, at Romanos etc., had description in 1992 (the same).

Control sequence can be also in the mRNA critical sequences of the increase genetic expression of the encoding sequence upstream of promotor downstream and gene (mRNA stabilizer) region.

The example in suitable mRNA critical sequences region is available from bacillus thuringiensis cryIIIA gene (WO94/25612) and subtilis SP82 gene (people such as Hue, 1995, Journal of Bacteriology177:3465-3471).

Control sequence can be also leader sequence, to the mRNA non-translational region by host cell translation outbalance.Leader sequence is operably connected to 5 ' end of the polynucleotide of coded polypeptide.Any leader sequence working in host cell all can be used.

For the suitable leader sequence of yeast host cell, derive from the gene of yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase, yeast saccharomyces cerevisiae alpha factor and yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

Control sequence can be also polyadenylation sequence, may be operably coupled to the sequence of 3 ' end of polynucleotide, and when transcribing, it is identified as the signal that adds polyadenylic acid residue to transcript mRNA by host cell.Any polyadenylation sequence working in host cell all can be used.

Useful polyadenylation sequence for yeast host cell is recorded in Guo and Sherman, 1995, Mol.Cellular Biol.15:5983-5990.

Control sequence can be also that coding is held the signal peptide being connected and guides polypeptide to enter the signal peptide coding region of emiocytosis path with polypeptide N.5 ' end of the encoding sequence of polynucleotide can be included in translation reading frame the signal coding sequence with the encoding sequence fragment Nature Link of coded polypeptide inherently.Or it is external signal coding sequence that 5 ' end of encoding sequence can comprise for encoding sequence.Encoding sequence is not natural while comprising signal coding sequence, extraneous signal peptide-coding sequence may need.Or for strengthening the secretion of polypeptide, extraneous signal peptide-coding sequence can replace natural signals peptide-coding sequence simply.Yet the signal coding sequence that the expressed polypeptide of any guiding enters the secretion path of host cell all can be used.

For the available signal peptide of yeast host cell, derive from the gene of yeast saccharomyces cerevisiae alpha factor and yeast saccharomyces cerevisiae saccharase.Other available signal coding sequences are recorded in the people such as Romanos, and 1992, the same.

Control sequence can be also the propeptide code sequence that coding is positioned at the propetide of polypeptide N end.The polypeptide obtaining is called proenzyme or propolypeptide (or being zymogen in some cases).Propolypeptide be generally inactivation and can be converted into active polypeptide by the catalysis from propolypeptide to propetide or autocatalysis cutting.Propeptide code sequence can derive from bacillus subtilis alkali proteinase (aprE), subtilis neutral protease (nprT), the thermophilic gene of ruining a bacterium (Myceliophthora thermophila) laccase (WO95/33836), rhizomucor miehei aspartic protease and yeast saccharomyces cerevisiae alpha factor.

When signal peptide sequence and propeptide sequence all exist, the N end of the N end of propeptide sequence next-door neighbour polypeptide and signal peptide sequence next-door neighbour propeptide sequence.

Also may need to add regulation and control polypeptide with respect to the regulating and controlling sequence of the expression of host cell growth.The example of regulator control system is in response to those of opening and closing that chemistry or physical stimulation cause genetic expression, comprises the existence of regulating compound.

Other examples of regulating and controlling sequence are to make those of gene amplification.In eukaryotic system, regulating and controlling sequence is included in the dihydrofolate reductase gene increasing under the existence of methotrexate and the metallothionein gene increasing under the existence of heavy metal.In these cases, the polynucleotide of coded polypeptide will operationally be connected with regulating and controlling sequence.

expression vector

The invention still further relates to and comprise polynucleotide of the present invention, promotor and transcribe the recombinant expression vector with translation termination signal.Various Nucleotide and control sequence can link together, and to produce the recombinant expression vector that can comprise one or more convenient restriction sites, this restriction site makes to insert or to replace in site the polynucleotide of coded polypeptide.Or, can in suitable expression vector, express polynucleotide by the nucleic acid construct that inserts polynucleotide or comprise polynucleotide.In the process of construction of expression vector, encoding sequence is arranged in carrier, thereby encoding sequence is operably connected by control sequence with suitable expression.

Recombinant expression vector can be any carrier (for example, plasmid or virus) that can carry out easily recombinant DNA program and can cause polynucleotide expression.The consistency of the host cell that carrier and carrier will import is depended in the selection of carrier conventionally.Carrier can be linear or closed cyclic plasmid.

Carrier can be self-replicating type carrier, as the outer entity of karyomit(e), exists and it copies the carrier that is independent of chromosome duplication, for example, plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier can comprise any element (means) of guaranteeing self-replacation.Or carrier can be, when importing host cell, be incorporated into genome in and with the carrier copying together with the karyomit(e) of its integration.In addition, can use single carrier or plasmid, or use two or more carriers or the plasmid that jointly comprises total DNA that will import in host cell gene group, or use transposon.

Carrier preferably comprises one or more allow to select easily transformant, transfectional cell, the isocellular selected marker things of transducer cell.Selected marker thing is following gene, and its product provides to the resistance of biocides or virus resistance, heavy metal resistance, with respect to auxotrophic prototroph etc.Appropriate flags thing for yeast host cell includes but not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

Preferred vector includes in the genome that makes carrier be incorporated into host cell or makes carrier in cell, be independent of genome and the element of self-replicating.

For being integrated into host cell gene group, carrier can depend on the polynucleotide sequence of coded polypeptide or through homologous recombination or non-homogeneous recombination and integration, enter any other element of genomic carrier.Or carrier can be integrated into other polynucleotide of host cell gene group containing being useful on guiding in chromosomal exact position via homologous recombination.The possibility of integrating for being increased in exact position, integrated element should comprise enough nucleic acid, for example 100～10,000 base pair, 400～10,000 base pair and 800～10,000 base pair, it has sequence identity highly with corresponding target sequence, to strengthen the possibility of homologous recombination.Integrated element can be with host cell gene group in any sequence of target sequence homology.In addition, integrated element can be the polynucleotide of noncoding polynucleotide or coding.On the other hand, carrier can be incorporated in the genome of host cell by non-homogeneous restructuring.

For self-replicating, carrier can also comprise make carrier can be in considered host cell the replication orgin of self-replicating.Replication orgin can be any plasmid replicon of the mediation self-replicating that works in cell.Term " replication orgin " or " plasmid replicon " are the polynucleotide of instigating plasmid or carrier to copy in vivo.

The example that is used in the replication orgin in yeast host cell is 2 microns of replication orgin ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

The polynucleotide of the present invention of more than one copy can be inserted in host cell, to increase polypeptide, produce.The increase of polynucleotide copies number can be by being integrated into the sequence of at least one additional copy in host cell gene group and obtaining; Or obtain by comprising can increase selectable marker gene and polynucleotide, like this, the cell of the selectable marker gene of amplification copy can be selected to contain by culturing cell under the existence of suitable selective agent, the cell of the polynucleotide of selecting thus to contain additional copy.

Be used for connecting said elements to build the step of recombinant expression vector of the present invention, be well known to those skilled in the art (referring to, for example, the people such as Sambrook, 1989, the same).

host cell

A second aspect of the present invention relates to defined yeast host cell in the step (a) as first aspect.

Preferably, the yeast host cell of this second aspect comprises at least one expression polynucleotide constructs of the present invention, and wherein protease-encoding gene is operably connected with one or more control sequences that instruct fusion polypeptide of the present invention to produce.The construct that comprises polynucleotide or carrier are imported in host cell, thereby make construct or carrier remain chromosomal integration or as the outer carrier of aforesaid self-replacation karyomit(e).Term " host cell " comprises the spawn of the parental cell that the sudden change because occurring in reproduction process is different from parental cell.

Host cell is yeast cell.Term used herein " yeast " comprises ascosporogenous yeast (Endomycetale), product load yeast and belongs to the yeast (gemma guiding principle) of imperfect fungi.Owing to may changing in future that is sorted in of yeast, for purposes of the present invention, yeast should be as Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc.App.Bacteriol.Symposium Series No.9,1980) in define as recording.

Yeast host cell can be mycocandida (Candida), Hansenula (Hansenula), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or Ye Shi yeast belong (Yarrowia) cell, Kluyveromyces lactis (Kluyveromyces lactis) for example, saccharomyces carlsbergensis (Saccharomyces carlsbergensis), yeast saccharomyces cerevisiae, saccharomyces diastaticus (Saccharomyces diastaticus), Saccharomyces douglasii, Crewe is yeast (Saccharomyces kluyveri) not, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica (Yarrowia lipolytica) cell.

Fungal cell can transform in a manner known way by relating to the method for protoplastis formation, protoplast transformation and cell walls regeneration.Yeast can be used the Guarente by Becker and, In Abelson, J.N.and Simon, M.I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume194, pp182-187, Academic Press, Inc., New York; The people such as Ito, 1983, J.Bacteriol.153:163; And the people such as Hinnen, the method that 1978, Proc.Natl.Acad.Sci.USA75:1920 records transforms.

Embodiment

definition

CDNA: term " cDNA " refers to the DNA molecular that can be come by the mRNA molecule preparation ripe, montage obtaining from eucaryon or prokaryotic cell prokaryocyte by reverse transcription.CDNA lacks the intron sequences that can be present in corresponding genomic dna.The precursor that initial elementary rna transcription thing is mRNA, it comprised by a series of that the step of montage processed before being rendered as mRNA ripe, montage.

Encoding sequence: term " encoding sequence " refers to the polynucleotide of the aminoacid sequence of direct appointment polypeptide.The border of encoding sequence determines by open reading frame conventionally, open reading frame with initiator codon as ATG, GTG or TTG starts and finish as TAA, TAG or TGA with terminator codon.Encoding sequence can be genomic dna, cDNA, synthetic DNA or its combination.

Control sequence: term " control sequence " refers to the essential nucleotide sequence that is expressed as for the polynucleotide of code book invention mature polypeptide.Each control sequence can be (native) (that is, from homologous genes) of interior life or external (foreign) (that is, from different genes) for the polynucleotide of coded polypeptide, or for being each other Nei Sheng or external.These control sequences include but not limited to, leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.Minimally, control sequence comprises promotor and transcribes and translation termination signal.For introducing the object of specific restriction enzyme site to promote that control sequence is connected with the coding region of the polynucleotide of coded polypeptide, control sequence can be provided with joint (linker).

Express: term " expressions " comprises related any step in polypeptide production, includes but not limited to: transcribe, post transcriptional modificaiton, translation, posttranslational modification and secretion.

Expression vector: term " expression vector " refers to and comprises the polynucleotide of coded polypeptide linearity or the ring-shaped DNA molecule being operably connected with control sequence for its expression.

Host cell: term " host cell " refers to that for the conversion of carrying out with the nucleic acid construct that comprises polynucleotide of the present invention or expression vector, transfection, transduction etc. be (susceptible) any cell type of susceptible.Term " host cell " comprises the spawn of the parental cell different from parental cell owing to undergoing mutation in reproduction process.

Separated: term " separated " refers to the form that occurs in non-natural or the material in environment.The limiting examples of separated material comprises: the material that (1) any non-natural occurs; (2) include but not limited to any material of any enzyme, variant, nucleic acid, albumen, peptide or cofactor, it shifts out at least in part from the composition to its natural relevant one or more or all natural appearance; (3) material with respect to natural appearance has manually modified any material; Or (4) by for example increasing material, with respect to (, the material encoding gene of a plurality of copies of the content to its natural other relevant components; Use the promotor stronger than the natural relevant promotor of gene to coding material) and the arbitrary substance of modification.Separated material may reside in fermented liquid sample.

Mature polypeptide: term " mature polypeptide " refers at translation and any posttranslational modification (holding brachymemma, glycosylation, phosphorylation etc. such as N end processing, the C) polypeptide in its final form afterwards.On the one hand, mature polypeptide is amino acid/11～412 of SEQ ID NO:2.

Mature polypeptide encoded sequence: term " mature polypeptide encoded sequence " refers to that coding has the polynucleotide of the mature polypeptide of enzymic activity.On the one hand, mature polypeptide encoded sequence is the Nucleotide 397～1632 of SEQ ID NO:1.

Fragment: term " fragment " for example means, from the amino acid whose polypeptide of the amino of mature polypeptide and/or carboxy terminal deletion one or more (, several); Wherein this fragment has retained its activity, for example, kept the proteolytic enzyme fragment of the protease activity of its derivative source full-length polypeptide.

The high stringent condition that waits: term " the high stringent condition that waits " refers to the probe at least 100 Nucleotide for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 50% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 70 ℃, each 15 minutes.

High stringent condition: the probe that it is at least 100 Nucleotide that term " high stringent condition " refers to for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 50% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 65 ℃, each 15 minutes.

In high stringent condition: the probe that it is at least 100 Nucleotide that term " in high stringent condition " refers to for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 35% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 60 ℃, each 15 minutes.

Medium stringent condition: the probe that it is at least 100 Nucleotide that term " medium stringent condition " refers to for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 35% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 55 ℃, each 15 minutes.

The low stringent condition that waits: term " the low stringent condition that waits " refers to the probe at least 100 Nucleotide for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 25% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 50 ℃, each 15 minutes.

The extremely low stringent condition that waits: term " the extremely low stringent condition that waits " refers to the probe at least 100 Nucleotide for length, at 42 ℃ 5 * SSPE, 0.3%SDS, 200 μ g/ml shear and the salmon sperm dna of sex change and 25% methane amide in prehybridization hybridization, follow standard Southern trace process and carry out 12～24 hours.Final 2 * SSC, the 0.2%SDS of using of solid support material cleans three times at 45 ℃, each 15 minutes.

Nucleic acid construct: term " nucleic acid construct " refers to from naturally occurring gene separated or be modified to and comprise strand nucleic acid fragment or synthetic or double-stranded nucleic acid molecule in the mode that can not occur in addition in nature, it comprises one or more control sequences.

Be operably connected: term " is operably connected " and refers to following structure, and wherein control sequence is placed in correct position place with respect to the encoding sequence of polynucleotide, thus the expression of control sequence guiding encoding sequence.

Sequence identity: the dependency between two aminoacid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use is at EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite (European molecular biology Freeware bag), the people such as Rice, 2000, Trends Genet.16:276-277) Needleman-Wunsch algorithm (the Needleman and Wunsch carrying out in the Needle program of (preferably 5.0.0 version or renewal), 1970, J.Mol.Biol.48:443-453) determine two sequence identity between aminoacid sequence.The optional parameter using: the open point penalty in room is 10, room expansion point penalty is 0.5, and EBLOSUM62 (the EMBOSS version of BLOSUM62) substitutes matrix.Be labeled as the Needle output of " the longest identity " (acquisition of use-nobrief option) as per-cent identity, be calculated as follows:

(identical residue * 100)/(the room sum in comparison length-comparison)

For purposes of the present invention, use is at EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite (European molecular biology Freeware bag), the people such as Rice, 2000, the same) Needleman-Wunsch algorithm (the Needleman and Wunsch that carries out in the Needle program of (preferably 5.0.0 version or renewal), 1970, the same) determine two sequence identity between deoxyribonucleotide sequence.The optional parameter using: the open point penalty in room is 10, room expansion point penalty is 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitutes matrix.Be labeled as the Needle output of " the longest identity " (acquisition of use-nobrief option) as per-cent identity, be calculated as follows:

(identical deoxynucleotide * 100)/(the room sum in comparison length-comparison)

Subsequence: term " subsequence " for example refers to, from the polynucleotide of 5 ' and/or 3 ' end disappearance of mature polypeptide encoded sequence one or more (, several) Nucleotide; Wherein subsequence coding has retained its active fragment.

Variant: term " variant " refers to the polypeptide with enzymic activity that for example, comprises change (replace, insert and/or lack) on one or more (, several) position.Replacement refers to that the aminoacid replacement with different occupies the amino acid of certain position; Disappearance refers to removes the amino acid that occupies certain position; And insert, refer to aminoacid addition to being also close to the amino acid that occupies certain position.

Embodiment

materials and methods

Bacterial strain:

Yeast saccharomyces cerevisiae YNG318:MATa Dpep4[cir+] ura3-52, leu2-D2, his4-539 is recorded in J.Biol.Chem.272 (15), and 9720-9727, in 1997.

Intestinal bacteria DH12S (purchased from Gibco BRL company) saves for yeast plasmid.

Plasmid:

All Yeast expression carriers are yeast saccharomyces cerevisiae and the shuttle vehicles under TPI promotor is controlled, and build the pJC039 describing in WO200192502.

Gene:

The synthetic pattern of the serine protease gene of the fierce fireball bacterium of archeobacteria is shown in SEQ ID NO:1.Coded mature amino acid sequence is shown in SEQ ID NO:2 (GeneSeqP:AAW2412), front 23 Amino acid profile secreting signal peptides wherein, and 109 amino acid following are that N end propetide and 110 final amino acid are C end propetide; Two propetides all become its activity form from express polypeptide cutting when maturation.

Coding is shown in SEQ ID NO:3 from the polynucleotide sequence of the signal sequence of Humicola insolens (Humicola insolens), and signal peptide is shown in SEQ ID NO:4 (GeneSeqP:AEE85970).

Use pGEM-T carrier system (Promega Corporation, Madison, WI, USA) glucoamylase gene from penicillium oxalicum (Penicillium oxalicum) is cloned into pGEM-T carrier (Promega Corporation, Madison, WI, US) in, to produce plasmid AMG1.By sequence verification, insert the AMG gene order of plasmid AMG1.The amino acid that the corresponding cDNA sequence of glucoamylase gene is shown in SEQ ID NO:5 and coded enzyme is shown in SEQ ID NO:6.

The phytase from the phytase gene coding of Peniophora (Peniophora lycii) CBS686.96 with GeneSeqP:AAW62858 aminoacid sequence; Corresponding wild-type cDNA sequence is shown in SEQ ID NO:7, and aminoacid sequence is shown in SEQ ID NO:8.

Coding is shown in SEQ ID NO:9 from the cDNA of the acid alpha-amylase of aspergillus niger (Aspergillus niger), and the aminoacid sequence of coding is shown in SEQ ID NO:10 (UNIPROT:P56271).

Initial separation is shown in SEQ ID NO:11 from the connector area of Luo Eratai bacterium (Athelia rolfsii), and the aminoacid sequence of coding is shown in SEQ ID NO:12.

Substratum and matrix:

-YPD:20g/L glucose, 20g/L peptone and 10g/L yeast extract.

-10 * base soln: 66.8g/l yeast nitrogen base w/o amino acid (DIFCO), 100g/l succinate and 60g/l NaOH.

-SC-glucose (or SC-substratum): 100ml/l20% glucose (that is, final concentration=2g/100ml of 2%), 4ml/l5% Threonine, 10ml/l1% tryptophane, 25ml/l20% casamino acids and 100ml/l10 * base soln.Use the strainer of 0.20 micron pore size to carry out sterilizing to this solution.By agar autoclaving together with H2O (about 761ml), and the SC-glucose solution of independent sterilizing is added in agar-agar soln.

-PEG/LiAc solution: 50ml40%PEG4000 (autoclaving) and 1ml5M Lithium Acetate (autoclaving).

-YPD: bacto peptone (Bacto peptone) 20g/l, yeast extract 10g/l, 20% glucose 100ml/l.

-zein (zein) agar plate: in 20mM sodium-acetate buffer (pH6), the zein of 0.05-0.1% (Sigma) and 2% agar.

DNA handles:

Except as otherwise noted, otherwise use as the people such as Sambrook (1989) Molecular cloning:A laboratory manual Cold Spring Harbor lab.Cold Spring Harbor, NY; Ausubel, the people such as F.M. (volume) " Current protocols in Molecular Biology ", John Wiley and Sons, 1995; Harwood, C.R. and Cutting, DNA manipulation and conversion are carried out in the molecular biological standard method of describing in S.M. (volume).

Yeast conversion:

For transformed yeast has utilized recombination mechanism in body, if carrier sequence and PCR fragment have identical flank region, by this mechanism yeast likely in vivo recombinant vectors sequence and PCR fragment to create expression vector.

By mixing carrier (HindIII-XbaI digestion) and the PCR fragment of 1 microlitre of 0.5 microlitre, prepare DNA mixture.Yeast saccharomyces cerevisiae YNG318 competent cell thaws on ice.In 12ml polypropylene tube, the cell of 100 microlitres is mixed with the carrier DNA of DNA mixture and 10 microlitres.Add 0.6ml PEG/LiAc solution and mix gently, then incubation 30 minutes under 30 ℃ and 200rpm.After this 42 ℃ (heat-shockeds), descend incubation 30 minutes, then transferred to eppendorf pipe also centrifugal 5 seconds.Remove supernatant liquor, and be dissolved in the YPD of 3ml.Then before being poured into SC-glucose flat board, by cell suspending liquid incubation 45 minutes at 200rpm and 30 ℃.

PCR reaction:

Except as otherwise noted, otherwise PCR reaction according to carrying out as follows.Comprise 48.5 microliters of water, the 100pmol/ microlitre primer of 0.5 microlitre, the PuReTaq Ready-To-Go of 0.5 microlitre template DNA and 2 pearls ^tMthe PCR reaction of PCR pearl (GE Healthcare) is carried out under the following conditions:

By Qiagen gel extraction kit, from sepharose, reclaim DNA fragmentation.The purifying fragment of gained is mixed with vector digestion thing.This mixing solutions is transformed for the yeast saccharomyces cerevisiae via recombinating in body.

Additive method:

The intestinal bacteria of rescue yeast plasmid transform by electroporation (BIO-RAD ^tMgene Pulser) carry out.

With plasmid kit prepare plasmid dna.DNA fragmentation passes through gel extraction kit reclaims from sepharose.

PCR is undertaken by PTC-200DNA Engine.

By ABI PRISM ^tM310 genetic analyzers are for measuring all DNA sequence dnas.

Yeast plasmid is prepared in a small amount test kit (Zymoprep Yeast Plasmid Minipreparation Kit) (Zymo Research) by Zymoprep yeast plasmid and is extracted.

Culturing yeast transformant in the 24 hole microtiter plates that contain YPD or shaking flask.

Proteolytic enzyme azo-casein is measured:

The substrate solution of the sample of 20 microlitres and 150 microlitres (ethanolic soln of 12.5% azo-casein of 4ml, in the 20mM of 96ml sodium-acetate, in pH4.5, contains 0.01%Triton-100) is mixed, and incubation 4 hours or longer time.Add after 100% trichoroacetic acid(TCA) (TCA) solution in 20 microlitres/hole, supernatant liquor plate is centrifugal and that pipette out 100 microlitres is measured A ₄₄₀.

Zein is dull and stereotyped to be measured:

By culture supernatants point sample, in zein agar plate, (0.1% zein, 100mM sodium-acetate buffer pH4) and at 70 ℃ are incubated overnight, to observe the transparent region via enzymatic reaction.

Phytase is dull and stereotyped to be measured:

By culture supernatants point sample, in phytate agar plate, (0.1% sodium phytate, 100mM sodium-acetate buffer pH4), and are incubated overnight at 37 ℃.By 0.5M CaCl ₂solution is above observed the transparent region via enzymatic reaction with precipitation phytic acid ca to dull and stereotyped.

Glucoamylase and α-amylase are dull and stereotyped to be measured:

By culture supernatants point sample, in Starch Agar flat board, (0.5% starch, 100mM sodium-acetate buffer pH4) and at 37 ℃ are incubated overnight, and then carry out iodine staining, observe the transparent region via enzymatic reaction.

the expression of embodiment 1. yeast saccharomyces cerevisiae bacterial serine in middle ancient times proteolytic enzyme

Primer pair PfuF (SEQ ID NO:13) and PfuR (SEQ ID NO:14) and Cuti-pfuF (SEQ ID NO:15) and PfuR (SEQ ID NO:14) amplification for the SEQ ID NO:1 synthetic gene of the archeobacteria fireball mycoproteinase of coding SEQ ID NO:2, wherein last group of primer pair has the natural construct of self signal sequence for increasing, then one group for build coding and N end from fierce fireball bacterium ripe before before the nucleotide sequence (being designated as cuti-PfuS) of the signal sequence from Humicola insolens at (SEQ ID NO:4) that merges of proteinase gene.

By by the PCR fragment obtaining together with being introduced in yeast saccharomyces cerevisiae YNG318 and transformed yeast with the pJC039 carrier of HindIII and XbaI and PvuII and XbaI digestion respectively.In the SC-agar glucose that contains 0.1% zein, cultivate the transformant obtaining.The results are shown in table 1; This cuti-PfuS transformant is because protease activity shows the faint halation around bacterium colony.With pre-PfuS, do not observe halation.

PfuF(SEQ?ID?NO:13):aacgacggtacccggggatcaagcttatgaaaggcctcaaggcattg

PfuR(SEQ?ID?NO:14):ctaattacatgatgcggccctctagattaggacgaaccaggctgc

cuti-pfuF(SEQ?ID?NO:15):gccttgttgctgctctccccgcgcctgagaaaaaggtgg

Table 1

?	Activity on zein flat board
		The fireball mycoproteinase with Humicola insolens at signal peptide	(+)
The fireball mycoproteinase with himself signal peptide	-

embodiment 2. is as the expression of the fireball mycoproteinase gene of fusion rotein

Zymoprotein and archeobacteria fireball mycoproteinase that construction of expression vector is expressed for good knitting, using and adopt the enzyme of good representation as carrier or traction apparatus albumen.

PCR reaction uses respectively primer pair PhyF (SEQ ID NO:16) and PhyR (SEQ ID NO:17), AMG-F (SEQ ID NO:18) and AMG-R (SEQ ID NO:19) and amyF (SEQ ID NO:20) and amyR (SEQ ID NO:21) to carry out, with the peniophora phytase gene that increases, penicillium oxalicum AMG gene and aspergillus niger acid alpha-amylase gene.Carrier enzyme gene is connected with the joint of proteinase gene via SEQ ID NO:11.

PhyF(SEQ?ID?NO:16):aacgacggtacccggggatcaagcttatggtttcttcggcattcg

PhyR(SEQ?ID?NO:17):tccacctttttctcaggcgcactacccgaggagccacccgggctt?gtagcaccttccgacggaacaaagc

AMG-F(SEQ?ID?NO:18):aacgacggtacccggggatcaagcttatgcgtctcactctattatc

AMG-R(SEQ?ID?NO:19):tccacctttttctcaggcgcactacccgaggagccacccgggc?ttgtagcaccacaagtggttgggactt

amyF(SEQ?ID?NO:20):aacgacggtacccggggatcaagcttatgagattatcgacttcgag?amyR(SEQ?ID?NO:21):tccacctttttctcaggcgcactacccgaggagccacccgggcttg?tagcacctcttccgctcccgccac

The PCR fragment of gained is mixed and is transformed in yeast saccharomyces cerevisiae with the plasmid cuti-PfuS through HindIII digestion respectively.In the SC-agar glucose that contains 0.1% zein, cultivate the transformant obtaining.All fusion protease transformants demonstrate with the construct only with proteinase gene and compare much bigger halation in periphery of bacterial colonies.

Also transformant is being contained at 27 ℃ in 24 orifice plates of 1ml YPD and cultivating 3 days, then testing the protease activity of supernatant liquor and other enzymic activity, described in method part.

These results show, when archeobacteria fireball mycoproteinase is expressed as fusion rotein, the expression level of this proteolytic enzyme in yeast has significantly and improve.

Table 2. symbol "-" means non-activity and can be detected; " (+) " means only to can be observed very faint active and " ++ ", and to mean enzymic activity high-visible.

Construct	Carrier enzyme	Protease activity	Carrier enzymic activity
				pro-PfuS	-	-	-
cuti-PfuS	-	(+)	-
				Phy-PfuS	Phytase	++	++
AMG-PfuS	Glucoamylase	++	++
				Amy-PfuS	α-amylase	++	++

Claims

1. in yeast, produce a method for proteolytic enzyme, comprising:

(a) provide the yeast host cell that comprises at least one polynucleotide expression construct, described polynucleotide expression construct coding translates with the second polypeptide the first polypeptide secreted by described host cell merging, and wherein said the second polypeptide is selected from:

(i) mature polypeptide of its maturing part and SEQ ID NO:2; Preferably have at least 60% with the position [1-412] of SEQ ID NO:2, for example the proteolytic enzyme of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity;

(ii) its maturing part is by the mature polypeptide encoded sequence with SEQ ID NO:1; Preferably have at least 60% with the position [397-1632] of SEQ ID NO:1, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or the coded proteolytic enzyme of polynucleotide of 100%DNA sequence identity;

(iii) its maturing part by low wait stringent condition, medium stringent condition, under high stringent condition, high stringent condition or the stringent condition such as high with the mature polypeptide encoded sequence of SEQ ID NO:1; Preferably with the position [397-1632] of SEQ ID NO:1; Or the coded proteolytic enzyme of polynucleotide of the total length complement hybridization of any one in both;

(iv) variant of the position [1-412] of the mature polypeptide of SEQ ID NO:2, preferred SEQ ID NO:2, it is included in one or more locational replacements, disappearance and/or insertion; With

(v) (i), (ii), (iii) or (iv) fragment with protease activity of polypeptide,

(b) under the condition of expression and secretion that is of value to described the first polypeptide, cultivate described host cell, secretion is translated fusion polypeptide and is produced ripe proteolytic enzyme thus; With

(c) optionally, reclaim proteolytic enzyme.

2. method according to claim 1, wherein said yeast is yeast belong, is preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).

3. method according to claim 1 and 2, wherein, described the first polypeptide is the enzyme of natural or allos total length or the brachymemma of C end, be preferably lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, aminopeptidase for example, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase, or xylobiase.

4. method according to claim 3, wherein said the first polypeptide is the glucoamylase of natural or allos total length or the brachymemma of C end; Be preferably the glucose starch enzyme polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

5. method according to claim 3, wherein said the first polypeptide is the phytase of natural or allos total length or the brachymemma of C end; Be preferably the phytase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

6. method according to claim 3, wherein said the first polypeptide is the α-amylase of natural or allos total length or the brachymemma of C end; Be preferably the α-amylase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

7. according to the method described in arbitrary aforementioned claim, wherein said at least one polynucleotide expression construct further comprises that coding and described the first polypeptide and described the second polypeptide translation merge and the 3rd polynucleotide of the connection peptides between them.

8. method according to claim 7, wherein said the 3rd polynucleotide encoding connection peptides; Preferably, described connection peptides comprises 2～200 amino acid; Preferably 3～100 amino acid, for example 4～50 or 5～25 amino acid; Most preferably, described connection peptides comprises the aminoacid sequence with SEQ ID NO:11 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

9. a yeast host cell, it comprises the polynucleotide expression construct of the first polypeptide of being secreted by described host cell that at least one coding and the second polypeptide translation are merged, wherein said the second polypeptide is selected from:

(i) mature polypeptide of its maturing part and SEQ ID NO:2 has at least 60%, for example the proteolytic enzyme of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity;

(ii) its maturing part is by having at least 60% with the mature polypeptide encoded sequence of SEQ ID NO:1, and for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or the coded proteolytic enzyme of polynucleotide of 100%DNA sequence identity;

(iii) its maturing part by low wait stringent condition, medium stringent condition, under high stringent condition, high stringent condition or the stringent condition such as high with the mature polypeptide encoded sequence of SEQ ID NO:1 or the coded proteolytic enzyme of the polynucleotide of its total length complement hybridization;

(iv) the misfolded proteins enzyme of the mature polypeptide of SEQ ID NO:2, it is included in one or more locational replacements, disappearance and/or insertion; With

(v) (i), (ii), (iii) or (iv) fragment with protease activity of polypeptide.

10. yeast host cell according to claim 9, it is yeast belong, is preferably yeast saccharomyces cerevisiae.

11. according to the yeast host cell described in claim 9 or 10, and wherein, described the first polypeptide is the enzyme of natural or allos total length or the brachymemma of C end, be preferably lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, aminopeptidase for example, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase, or xylobiase.

12. yeast host cells according to claim 11, wherein said the first polypeptide is the glucoamylase of natural or allos total length or the brachymemma of C end; Be preferably the glucose starch enzyme polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

13. yeast host cells according to claim 11, wherein said the first polypeptide is the phytase of natural or allos total length or the brachymemma of C end; Be preferably the phytase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

14. yeast host cells according to claim 11, wherein said the first polypeptide is the α-amylase of natural or allos total length or the brachymemma of C end; Be preferably the α-amylase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

15. according to the yeast host cell described in arbitrary aforementioned claim, and wherein said at least one polynucleotide expression construct further comprises that coding and described the first polypeptide and described the second polypeptide translation merge and the 3rd polynucleotide of the connection peptides between them.

16. yeast host cells according to claim 15, wherein said the 3rd polynucleotide encoding connection peptides; Preferably, described connection peptides comprises 2～200 amino acid; Preferably 3～100 amino acid, for example 4～50 or 5～25 amino acid; Most preferably, described connection peptides comprises the aminoacid sequence with SEQ IDNO:11 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

17. 1 kinds of polynucleotide expression construct, its coding translates with the second polypeptide the first polypeptide secreted by host cell merging, and wherein said the second polypeptide is selected from:

(v) (i), (ii), (iii) or (iv) fragment with protease activity of polypeptide.

18. polynucleotide expression construct according to claim 17, wherein, described the first polypeptide is the enzyme of natural or allos total length or the brachymemma of C end, be preferably lytic enzyme, isomerase, ligase enzyme, lyase, oxydo-reductase or transferring enzyme, aminopeptidase for example, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, become glycanase, oxydase, pectin decomposing enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase, or xylobiase.

19. polynucleotide expression construct according to claim 18, wherein said the first polypeptide is the glucoamylase of natural or allos total length or the brachymemma of C end; Be preferably the glucose starch enzyme polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

20. polynucleotide expression construct according to claim 18, wherein said the first polypeptide is the phytase of natural or allos total length or the brachymemma of C end; Be preferably the phytase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

21. polynucleotide expression construct according to claim 18, wherein said the first polypeptide is the α-amylase of natural or allos total length or the brachymemma of C end; Be preferably the α-amylase polypeptide that is selected from natural or allos following total length or the brachymemma of C end:

22. according to the polynucleotide expression construct described in arbitrary aforementioned claim, and wherein said at least one polynucleotide expression construct further comprises that coding and described the first polypeptide and described the second polypeptide translation merge and the 3rd polynucleotide of the connection peptides between them.

23. polynucleotide expression construct according to claim 22, wherein said the 3rd polynucleotide encoding connection peptides; Preferably, described connection peptides comprises 2～200 amino acid; Preferably 3～100 amino acid, for example 4～50 or 5～25 amino acid; Most preferably, described connection peptides comprises the aminoacid sequence with SEQ ID NO:11 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.