CN1332802A - Production of recombinant monellin using methylotrophic yeast expression system - Google Patents

Abstract

The present invention relates to a single-chain monellin-like protein which is stable and which is at least 100-fold sweet as compared to sucrose on the weight basis. The present invention also relates to a nucleic acid encoding said monellin-like protein. Preferably, the nucleic acid further comprises a promoter and a signal sequence for directing expression and secretion of the encoded monellin-like protein in the methylothrophic yeast Pichia pastoris. The present invention further relates to a recombinant Pichia pastoris cell containing the nucleic acid encoding the monellin-like protein, a process for producing the monellin-like protein from the recombinant Pichia pastoris and product of the process.

Description

With methylotrophy yeast expression system production reorganization Mo Neilin

Require the right of priority of following application according to United States Code 35 § 119 (e) the application: applicant Lingxun Duan, U.S. Provisional Application sequence number 60/114,529, on December 31 1998 applying date, name is called with methylotrophy yeast expression system production reorganization Mo Neilin.

1. invention field

The present invention relates to the Mo Neilin sample albumen of a strand, this albumen is stable, and is than sucrose sweet 100 times at least on basis with weight.The proteic nucleic acid of described Mo Neilin sample the present invention also relates to encode.Preferably, this nucleic acid further comprises a promotor and a signal sequence, and this sequence guides the proteic expression of Mo Neilin sample and the secretion of coding in methylotrophy yeast Pichia pastoris.The invention further relates to a reorganization Pichiapastoris cell that contains the proteic nucleic acid of coding Mo Neilin sample, a kind of product of from reorganization Pichia pastoris, producing the proteic method of Mo Neilin sample and this method.

2. background technology

2.1. Mo Neilin

Mo Neilin belong to high monellin family from tropical plants (Dansby, Nature Biotechnol is learned, 1997,15:419-420).Sweet about 3,000 times of Mo Neilin than sucrose.Other similar albumen comprises thaumatin, miraculin, mabinlin, pentadin and aspartame (Id.).Mo Neilin is isolating (United States Patent (USP) the 3rd, 878,184 and 3,998, No. 798 from the plant Dioscorephyllum comminisii of West Africa at first; Morris and Cagan, Biochim.Biophys.Acta, 1972,261:114-122).The aminoacid sequence of Mo Neilin, three-dimensional structure and various physics and chemical property are determined (Ogata etc., nature, 1987.328:739-742; Morris etc., journal of biological chemistry, 1973.248:534-539; Cagan, science, 1973,181:32-35; Bohak and Li, Biochim.Biophys.Acta, 1976,427:153-170; Hudson and Beeman, Biochem.Biophys.Res.Comm., 1976,71:212-220; Van der Wel and Loeve, FEBS Lett., 1973,29:181-183; With Frank and Zuber, HoppeSeyler ' s Z Physiol.Chem., 1976,357:585-592).

United States Patent (USP) the 4th, 300 discloses the sootiness particle that contains thaumatin or Mo Neilin No. 576.United States Patent (USP) the 4th, 562 discloses bag for No. 076 by the chewing gum of thaumatin or Mo Neilin.United States Patent (USP) the 4th, 412, disclose for No. 984 contain thaumatin or Mo Neilin add flavor synergic oral compositions.Yet, although it has the advantage of low-calorie sweeteners, but since it to heat and the unstable of pH, obtain the shortage of supplying plant origin and the uncertainty of dispensing mode during as foodstuff additive, make that commercial applications is subjected to hindering (Dansby widely, NatureBiotechnology, 1997,15:419-420).

1989, Sung-Hou Kim study group report by genetically engineered in intestinal bacteria, produce strand Mo Neilin (Kim etc., protein engineering 1989,2:571-575).The strand Mo Neilin that finds purifying is more stable and can tolerate the pH scope of broad to heat, and has kept sugariness.Several aspects of the present invention have become the theme of some United States Patent (USP).For example, United States Patent (USP) the 5th, 234 discloses the formation of expressing strand Mo Neilin in vegetable cell for No. 834.United States Patent (USP) the 5th, 487, No. 923 disclose molecular formula is the monellin compound of B-C-A, wherein B represents a peptide moiety, has 90% identity at least with the 1-46 residue of natural Mo Neilin B chain and only by conservative substituting group modification; C is a covalent linkage, or one hydrophilic, physiology is acceptable, the covalently bound unit that is equivalent to 1-10 amino acid peptide space length can be provided, and selected amino acid resides in the outside of molecule and do not disturb natural configuration; A represents the 6-45 residue at least 90% with natural Mo Neilin A chain consistent also only by guarding the peptide that substituting group is modified.United States Patent (USP) the 5th, 487 discloses an expression system of making the 5th, 487, No. 923 disclosed strand Mo Neilin of United States Patent (USP) for No. 983.United States Patent (USP) the 5th, 670 discloses the DNA of the 5th, 487, No. 923 disclosed strand Mo Neilin of coding United States Patent (USP) No. 339.United States Patent (USP) the 5th, 672 discloses the method that increases food mixture sweet tastes with the 5th, 487, No. 923 disclosed strand Mo Neilin of United States Patent (USP) for No. 372.United States Patent (USP) the 5th, 264 discloses a strand Mo Neilin albumen, in the taste test of standard, has been that unit is 50 times of sucrose at least with weight for No. 558.

Recently, Kondo etc., Nature Biotechnol is learned, and 1997,15:453-457 discloses the proteic heterogenous expression of strand Mo Neilin in beads belongs to yeast cell.But its report Mo Neilin high level expression, stabilize proteins account for＞and 50%.

2.2. the expression of heterologous protein in PICHIA PASTORIS

Methylotrophy yeast Pichia pastoris has been used as protein expression system.Several aspects of this expression system have become the theme of some United States Patent (USP).As, United States Patent (USP) the 4th, 837, the autonomously replicating sequence that discloses Pichia pastoris No. 148.United States Patent (USP) the 4th, 855 discloses the control region of allogeneic gene expression in Pichia pastoris cell No. 231.United States Patent (USP) the 4th, 882, the site selectivity genomic modification that discloses Pichia pastoris No. 279.United States Patent (USP) the 4th, 929 discloses for No. 555 and to make the full cell of Pichia pastoris be in competence so that the method that transforms.United States Patent (USP) the 5th, 122 discloses for No. 465 and produced the step that can select phenotype in Pichia pastoris bacterial strain.United States Patent (USP) the 5th, 324 discloses at the methylotrophy cell, has comprised in the Pichia pastoris cell production of insulin-like growth factor-i for No. 639.

Several signal sequences have been used to guide the secretion at the heterologous protein of Pichia pastoris cell inner expression.The example of these signal sequences includes but not limited to, the signal sequence of Pichia pastoris acid phosphatase, the signal sequence of Aspergillus giganteus α-Zhou Qujunsu (Martinez-Ruiz etc., Protein Expr.Purif., 1998,12 (3): 315-22; Abdulaev etc., ProteinExpr.Purif., 1997,10 (1): 61-9; Kotake etc., J.Lipid Res., 1996,37 (3): 599-605), signal sequence (the alphaNAGAL of α-t-N-acetylgalactosamine enzyme, EC 3.2.1.49) (Zhu etc., Arch.Biochem.Biophys., 1998,352 (1): 1-8), proteic signal peptide (the Heim etc. of OmpA, Biochim.Biophys.Acta., 1998,1396 (3): 306-19), in the signal sequence of mouse α-factor signal (cCel1) or the pepper-β-1, natural signals sequence (the Ferrarese etc. of 4-dextranase, FEBS Lett., 1998,422 (1): 23-6), signal peptide (the Jonsson etc. of isolating laccase from wooden dissolved mould Trametes, Curr Genet., 1997,32 (6): 425-30), signal peptide (the Merkle etc. of mouse lysosomal acid alpha-Mannosidase, Biochim.Biophys.Acta., 1997,1336 (2): 132-46), signal peptide (the Wuebbens etc. of pig carbonic anhydrase inhibitor, biological chemistry, 1997,36 (14): 4327-36), signal sequence (the Fierobe etc. of awamori aspergillus tubigensis glucoamylase, Protein Expr.Purif, 1997,9 (2): 159-70), signal sequence (the Ferrari etc. of the main urine protein of mouse, FEBS Lett, 1997,401 (1): 73-7), signal sequence (the Skory etc. of pho 1, Curr.Genet., 1996,30 (5): 417-22), signal sequence (the Sadhukhan etc. of rabbit Zinc metallopeptidase Zace1 (ACE), journal of biological chemistry, 1996,271 (31): 18310-3), propeptide sequence (the Tsujikawa etc. of Pichia pastoris aspartate protease, yeast, 1996,12 (6): 541-53), signal sequence (the Ohi etc. of Pichia pastorisPRCl, yeast, 1996,12 (1): 31-40), the signal sequence of the thermally-stabilised α-Dian Fenmei of bacterium and from the SUC2 gene signal sequence (Paifer etc. of yeast saccharomyces cerevisiae, yeast, 1994,10 (11): 1415-9) and signal sequence (Fidler etc., the J.Mol.Endocrinol. of yeast saccharomyces cerevisiae hybridization pheromone α-factor, 1998,21 (3): 327-336).

Although methylotrophy yeast Pichia pastoris has been successfully used to produce various heterologous proteins, but United States Patent (USP) the 5th, 324, point out for No. 639,, be difficult to the given gene of prediction and whether can in this primary yeast, express whether can tolerate and have the recombination product in its cell the understanding level of methylotrophy yeast expression system with at present to proper level or this yeast host.United States Patent (USP) the 5th, 324 is further pointed out for No. 639, and difficulty is whether specific protein of prediction can be secreted by the methylotrophy yeast host especially, and if can secrete, with what efficient secretion.For example, Vollmer etc., immunological method magazine, 1996,199 (1): when 47-54, report are suitable for the expression of methylotrophy pichia pastoris/secretion vector when 323 amino-acid residues insertions of people sIL-6R, do not observe Recombinant Protein Expression and the secretion that to measure.So far, Mo Neilinshang does not express with the Pichiapastoris expression system and secreted.

Based on the strong interest of commercial applications Mo Neilin, press for more efficient methods to produce stable Mo Neilin, it still keeps its natural sugariness and simplifies the purification step in downstream.The present invention is conceived to these and other demands in this area.The document of quoting on this should not be interpreted as admitting that these literature contents are prior aries of the present invention.

3. summary of the invention

The present invention relates to an isolating nucleic acid, this nucleic acid comprises the nucleotide sequence of a coding chimeric protein, described chimeric protein comprises from the N-terminal to the C-terminal: a) the first peptidyl fragment, it is by form with the aminoacid sequence of 1-50 residue 40% identity of natural Mo Neilin B chain at least, wherein hundred identity proportions by subtraction be with the aminoacid sequence of the identical size of natural Mo Neilin B chain on determine; B) peptide base key, or the second peptidyl fragment, it is made up of 1-12 amino acid; And c) aminoacid sequence of 1-45 residue at least 40% identity of the natural Mo Neilin A of tripeptides substrate Duan Youyu chain is formed, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin A chain on determine, wherein said chimera protein is than equivalent sucrose sweet 100 times at least of stable and a certain amount of described chimera proteins, in described nucleic acid, use the codon that is preferably used by yeast cell.Preferably, isolating nucleic acid further one of coding can be in Pichia pastoris the pilot protein expression promoter, and/or can guide coded chimera protein excretory aminoacid sequence from Pichia pastoris.

The present invention also relates to a reorganization Pichia pastoris cell that contains above-mentioned nucleic acid.The invention further relates to the method for production chimera protein, comprise that cultivating the reorganization Pichiapastoris cell that contains above-mentioned nucleic acid reclaims expression and excretory chimera protein so that the chimera protein of coding by cell expressing and secretion, reaches.At last, the present invention relates to the product of aforesaid method.

4. brief description of drawings

Figure l shows proteic aminoacid sequence of recombinant single chain Mo Neilin and the proteic dna sequence dna of coding recombinant single chain Mo Neilin.Amino-acid residue 1-50 is equivalent to the 1-50 amino-acid residue of natural Mo Neilin B chain; Amino-acid residue 51 is the glycine as connector element; Amino-acid residue 52-96 is equivalent to the 1-45 amino-acid residue of natural Mo Neilin A chain.

Fig. 2 demonstration is used for the synthetic proteic DNA oligomer of recombinant single chain Mo Neilin sequence.

Fig. 3 shows the position of each DNA oligomer in synthetic Mo Neilin DNA and its enzymic digestion site.

Fig. 4 shows the estriction map of reorganization Mo Neilin protein expression vector pGWYS1.

Fig. 5 shows the structure of pGWYS1.

Fig. 6 shows that the proteic SDS-PAGE of isolating excretory reorganization Mo Neilin analyzes from substratum.

Fig. 7 shows the proteic purification step of excretory reorganization Mo Neilin.

5. detailed description of the present invention

The invention provides the nucleic acid of a coding strand Mo Neilin sample albumen, this albumen is stable and take weight as the basis than sucrose sweet 100 times at least. Preferably, this nucleic acid further comprises a promoter and burst, with Mo Neilin sample protein expression and the secretion of guiding coding in methylotrophic yeast Pichia pastoris. The present invention also provides a restructuring Pichia pastoris cell that contains the nucleic acid of coding Mo Neilin sample albumen, produces the method for Mo Neilin sample albumen and the product of the method by restructuring Pichia pastoris.

For illustrating the disclosure, but not as restriction, detailed description of the present invention is divided into following portions.

5.1. the proteic nucleic acid of coding strand Mo Neilin

The invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, described chimera protein comprises from the N-terminal to the C-terminal: a) by first peptidyl fragment of forming with the aminoacid sequence of 1-50 residue at least 40% identity of natural Mo Neilin B chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin B chain on determine; B) peptide base key, or second peptidyl fragment forming by 1-12 amino acid; And c) by the 3rd the peptidyl fragment of forming with the aminoacid sequence of 1-45 residue at least 40% identity of natural Mo Neilin A chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin A chain on determine, wherein said chimera protein is than equivalent sucrose sweet 100 times at least of stable and a certain amount of described chimera proteins, in described nucleic acid, use the codon that is preferably used by yeast cell.

In a specific embodiment, the invention provides the isolating nucleic acid of the nucleotide sequence that comprises the chimera protein of encoding, wherein first peptidyl fragment is by forming with the aminoacid sequence of natural Mo Neilin B chain at least 60% identity.Preferably, first peptidyl fragment is by forming with the aminoacid sequence of natural Mo Neilin B chain at least 90% identity.More preferably, first peptidyl fragment is made up of the 1-50 amino-acid residue of natural Mo Neilin B chain.

In another specific embodiment, the invention provides the isolating nucleic acid of the nucleotide sequence that comprises the chimera protein of encoding, wherein second peptidyl fragment is made up of aminoacid sequence Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser (SEQ ID NO:1).Preferably, second peptidyl fragment is made up of aminoacid sequence Gly-Gly-Gly-Ser (SEQ ID NO:2).More preferably, second peptidyl fragment is made up of amino-acid residue Gyl.

Still in another specific embodiment, the invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, wherein the 3rd peptidyl fragment is by forming with the aminoacid sequence of natural Mo Neilin A chain at least 60% identity.Preferably, the 3rd peptidyl fragment is by forming with the aminoacid sequence of natural Mo Neilin A chain at least 90% identity.More preferably, the 3rd peptidyl fragment is made up of the 1-45 amino-acid residue of natural Mo Neilin A chain.

In a preferred embodiment, the invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, wherein first peptidyl fragment is made up of the l-50 amino-acid residue of natural Mo Neilin B chain, second peptidyl fragment is made up of amino-acid residue Gly, and the 3rd peptidyl fragment is made up of the 1-45 amino-acid residue of natural Mo Neilin A chain.

In a specific embodiment, the invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, this albumen can be by anti-Mo Neilin or the combination corresponsively of anti-thaumatin antibody mediated immunity.

In another specific embodiment, the invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, wherein chimera protein further comprises bootable described chimera protein excretory aminoacid sequence from Pichia pastoris.Preferably, the secretion homing sequence is the endogenous signal sequence of Pichia pastoris.More preferably, the endogenous signal sequence is selected from Pichia pastoris acid phosphatase, Pichia pastoris aspartate protease and by the signal sequence of the Pichia pastoris carboxypeptidase y of Pichia pastoris PRC1 coding.In addition selectively, the secretion homing sequence is a yeast signal sequence, and wherein said yeast is not Pichia pastoris.Preferably, the yeast signal sequence is the signal sequence from yeast saccharomyces cerevisiae.More preferably, the yeast saccharomyces cerevisiae signal sequence is selected from the signal sequence of Saccharomyces Cerevisiae in S UC2 and yeast saccharomyces cerevisiae hybridization pheromone α-factor.Most preferably, the yeast saccharomyces cerevisiae signal sequence is the signal sequence of yeast saccharomyces cerevisiae hybridization pheromone α-factor.Other can be used for secretion homing sequence of the present invention and includes but not limited to, in the Aspergillus giganteus α-Zhou Qujunsu, alpha-N-Acetylgalactosaminidase, OmpA albumen, mouse α-factor (cCel1), pepper-and β-1,4-dextranase, the signal sequence of isolating laccase, mouse lysosomal acid alpha-Mannosidase, pig carbonic anhydrase inhibitor, awamori aspergillus tubigensis glucoamylase, the main urine protein of mouse, pho1, rabbit Zinc metallopeptidase Zace1 (ACE) and the thermally-stabilised α-Dian Fenmei of bacterium from molten wooden fungi Trametes.

In a specific embodiment, the invention provides an isolating nucleic acid that comprises the nucleotide sequence of the chimera protein of encoding, this nucleic acid is DNA.In another specific embodiment, the invention provides an isolating nucleic acid, it can be hybridized with the dna sequence dna of coding chimera protein.Still in another certain embodiments, the invention provides an isolating nucleic acid with the nucleotide sequence complementary nucleotide sequence that comprises the chimera protein of encoding.

In a certain embodiments, the invention provides the DNA of a coding chimera protein, this DNA further comprise one can be in Pichia pastoris the pilot protein expression promoter.Preferably, this promotor is the endogenous promotor of Pichia pastoris.More preferably, the endogenous promotor is the promotor of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP).In addition, though be not preferred, also can use the promotor of methyl alcohol response gene in the methylotrophy yeast.The example that this methyl alcohol is replied promotor includes but not limited to, from the promotor of the one-level alcohol oxidase gene of Pichiapastoris AOX1, from the promotor of the secondary alcohol oxidase gene of Pichia pastorisAOX2, from the promotor of the Protosol synthase gene of Pichia pastoris (DAS), from the promotor of the P40 gene of Pichia pastoris, from the promotor of the catalase gene of Pichia pastoris, Deng (seeing United States Patent (USP) the 5th, 324, No. 639).

In another specific embodiment, the invention provides the DNA of a coding chimera protein, this DNA further comprises the sequence that makes it duplicate and select in bacterium.In this way, a large amount of dna fragmentations can be produced by duplicating in the bacterium.

In a preferred embodiment, the invention provides the DNA of a coding chimeric protein, wherein in the chimera protein of coding, first peptidyl fragment is made up of the 1-50 amino-acid residue of natural Mo Neilin B chain, second peptidyl fragment is made up of amino-acid residue Gly, the 3rd peptidyl fragment is made up of the 1-45 amino-acid residue of natural Mo Neilin A chain, and described DNA further comprises the signal sequence of promotor and the yeast saccharomyces cerevisiae hybridization pheromone α-factor of Pichia pastoris GAP.

In another preferred embodiment, the invention provides the DNA of a coding chimera protein, wherein use the codon that is preferably used by Pichia pastoris cell.

In a most preferred embodiment, the invention provides the DNA of a coding chimera protein, wherein dna molecular comprises nucleotide sequence or the dna vector as describing among Fig. 4 as describing among Fig. 1.

The nucleic acid that comprises the nucleotide sequence of the chimera protein disclosed herein of encoding, or its any fragment, analogue or derivatives thereof, available any method known in the art obtains.Nucleic acid can be by chemosynthesis entirely.Selectively be in addition, coding chimera protein each fragment, promptly first, second or the segmental nucleic acid of tripeptides base can obtain by molecular cloning, or in the required cell purifying.Then, coding chimera protein each segmental nucleic acid can by chemically or the enzyme connection link together, comprise the nucleic acid of the nucleotide sequence of the chimera protein disclosed herein of encoding with formation, or its any fragment, analogue or derivatives thereof.

Any Dioscoreophyllum comminisii cell can be used as the source of nucleic acid potentially to separate the nucleic acid of coding Mo Neilin.Selectively be that the nucleic acid of coding Mo Neilin can be according to natural Mo Neilin aminoacid sequence design and synthetic (also the seeing United States Patent (USP) the 5th, 478, No. 923) described among Fig. 1 in addition.

DNA can clone by chemosynthesis, cDNA from clone's DNA (as DNA " library ") or obtain by genomic dna or its segmental clone with the method for standard known in the art, purifying (is seen from needed cell, for example, Sambrook etc., 1989, molecular cloning, lab guide, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (volume), 1985, dna clone: research practice, MRL Press, Ltd., Oxford, U.K.Vol.I, II.) clone who derives from genomic dna can contain regulation and control and introne DNA zone except that the codon zone; Clone from cDNA should only contain exon sequence.No matter it is originated, and gene answers molecule ground clone to advance a suitable carrier to propagate gene.

From the gene molecule clone in cDNA source, cDNA produces from total cell RNA or mRNA with method well known in the art.Gene also can obtain from genomic dna, when producing dna fragmentation (as with Restriction Enzyme or pass through mechanical shearing), the gene that the some of them codified is required.Linear DNA fragment can adopt standard techniques to separate according to size then, includes but not limited to agarose and polyacrylamide gel electrophoresis and column chromatography.

In case comprise that the nucleic acid of nucleotide sequence of the chimera protein disclosed herein of encoding or its any fragment, analogue or derivatives thereof are obtained, its identity can pass through that nucleic acid sequencing (with any method well known in the art) is determined and with known sequences relatively.Dna sequence analysis can carry out with any technology known in the art, include but not limited to method (Maxam and the Gilbert of Maxam and Gilbert, 1980, Meth.Enzymol., 65:499-560), two method of deoxidation (Sanger etc., 1977 of Sanger, Proc.Natl.Acad.Sci.U.S.A., 74:5463), use T7 archaeal dna polymerase (Tabor and Richardson, United States Patent (USP) the 4th, 795, No. 699), use an automated DNA sequenator (as Applied Biosystems, Foster City, or the method for describing among the PCT publication WO97/15690 CA).

Nucleic acid with the nucleic acid of the nucleotide sequence that comprises the chimera protein disclosed herein of encoding or its any fragment, the hybridization of analogue or derivatives thereof, can be under the condition of low, height or moderate strictness separate and (also see Shilo and Weinberg by nucleic acid hybridization, 1981, Proc.Natl.Acad.Sci.USA, 78:6789-6792).

As used herein, the strand Mo Neilin chimera protein that " stablizing " is meant statement is placing about 4 ℃ to keep its sugariness of at least 70% after at least 5 minutes at least 2.5 hours or about 100 ℃ at least 6 months or about 60 ℃.In addition, " stablizing " the strand Mo Neilin chimera protein that is meant statement is placing the about 2.0-of pH scope to keep its sugariness of at least 70% at least after about 11.0 6 hours.

The sugariness of the strand Mo Neilin chimera protein of statement can be measured with common taste test known in the art.For example, on weight basis by the sugariness (also see United States Patent (USP) 5th, 478, No. 923) of appropriate thinning ratio than sucrose.

Preferably the codon that is utilized by yeast cell can be measured with methods known in the art, as in Sharp etc., nucleic acids research, 1986,14 (13): 5125-43 and Li and Luo, J.Theor.Biol., 1996,181 (2): disclosed method among the 111-24.According to the method for Sharp, the key character of preferred codon comprises that deviation and less A+T base pair are inclined to largely for, three base pyrimidine relevant with the many degree of tRNA in the yeast.Li and Luo disclose the method for gene expression dose in classification and prediction intestinal bacteria and the yeast cell, and it is called the information cluster (SCIC) of self-consistent.Adopt the codon of improvement to adopt index (CAI) value, Li and Luo finished to based composition in intestinal bacteria and the yeast, base is relevant and gene expression dose between the linear regression analysis that concerns.Li and Luo have also proposed to express the hypothesis that strengthens network site (EENS), and the linear equation between the existence of this hypothesis is can be by base in genetic expression and the codon, in the adjacent codon and in the non-adjacent codon relevant shows.In addition, can use and be successfully used to the proteic codon of expressing heterologous in Pichia pastoris cell.The example of these codons is found in United States Patent (USP) the 4th, 837, No. 148; United States Patent (USP) the 4th, 855, No. 231; United States Patent (USP) the 4th, 882, No. 279; United States Patent (USP) the 4th, 929, No. 555; United States Patent (USP) the 5th, 122, No. 465; United States Patent (USP) the 5th, 324, No. 639; Martinez-Ruiz etc., Protein Expr.Purif., 1998,12 (3): 315-22; Abdulaev etc., the really good Purif. of Protein Expr., 1997,10 (1): 61-9; Kotake etc., J.Lipid Res., 1996,37 (3): 599-605; Zhu etc., Arch.Biochem.Biophys., 1998.352 (1): 1-8; Heim etc., Biochim.Biophys.Acta., 1998,1396 (3): 306-19; Ferrarese etc., FEBS Lett., 1998,422 (1): 23-6; Jonsson etc., Curr.Geneet, 1997,32 (6): 425-30; Merkle etc., Biochim.Biophys.Acta., 1997,1336 (2): 132-46; Wuebbens etc., biological chemistry, 1997,36 (14): 4327-36; Fierobe etc., Protein Expr.Purif., 1997,9 (2): 159-70; Ferrari etc., FEBS Lett., 1997,401 (1): 73-7; Skory etc., Curr.Genet., 1996,30 (5): 417-22; Sadhukhan etc., resultant The Chemicals, 1996,271 (31); 18310-3; Tsujikawa etc., yeast, 1996,12 (6); 541-53; Ohi etc., yeast, 1996,12 (1): 31-40; Paifer etc., yeast, 1994,10 (11): 1415-9; Fidler etc., J.Mol.Endocrinol., 1998,21 (3): 327-336; With Brocca etc., albumen science, 1998,7 (6): 1415-22.

Whether a chimera protein can be determined in conjunction with available methods known in the art corresponsively by anti-Mo Neilin or anti-thaumatin antibody mediated immunity.The example that can be used for anti-Mo Neilin of the present invention or anti-thaumatin antibody includes but not limited to following public antibody, Slootstra etc., Chem.Senses, 1995.20 (5): 535-43; Antonenko and Zanetti, life science, 1994,55 (15): 187-92; Bodani etc., hybridoma, 1993,12 (2): 177-83; Mandal etc., hybridoma, 1991,10 (4): 459-66 and Haimovich, Isr.J.Med.Sci., 1975,11 (11): 1183.

5.2. the proteic generation of Mo Neilin from reorganization PICHIA PASTORIS cell

In a specific embodiment, the invention provides a reorganization Pichia pastoris cell that contains the nucleic acid of the chimera protein of encoding, described chimera protein comprises from the N-terminal to the C-terminal: a) by first peptidyl fragment of forming with the aminoacid sequence of 1-50 residue at least 40% identity of natural Mo Neilin B chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin B chain on determine; B) peptide base key, or second peptidyl fragment forming by 1-12 amino acid; And c) by the 3rd the peptidyl fragment of forming with the aminoacid sequence of 1-45 residue at least 40% identity of natural Mo Neilin A chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin A chain on determine, wherein said chimera protein is that stable and a certain amount of described chimera protein is that 100 times of equivalent sucrose are sweet at least, in the described therein nucleic acid, use the codon that is preferably used by yeast cell.Preferably, reorganization Pichia pastoris cell contains a dna molecular that comprises the dna vector of describing among the nucleotide sequence described among Fig. 1 or Fig. 4.The reorganization Pichia pastoris cell that comprises disclosed nucleic acid in 4.1 joints also is provided.

Transform the method for methylotrophy yeast such as Pichia pastoris, the method that reaches applicable cultivation methylotrophy yeast cell is that this area is known usually, contains the gene of the heterologous protein of encoding in the genome of this yeast cell.Preferably, be disclosed in United States Patent (USP) the 4th, 837, No. 148, No. the 4th, 855,231, United States Patent (USP), United States Patent (USP) the 4th, 882, No. 279, No. the 4th, 929,555, United States Patent (USP), United States Patent (USP) the 5th, 122, the conversion that No. the 5th, 324,639, No. 465 and United States Patent (USP), positive conversion product select and cultural method can be used for the present invention.

In another specific embodiment, the invention provides a kind of method of the Mo Neilin of generation chimera protein, comprise and cultivate the reorganization Pichia pastoris cell that contains the disclosed nucleic acid of 4.1 joints, make the chimera protein of this cell expressing and secretion coding, and reclaim and express and the excretory chimera protein.Preferably, use the reorganization Pichia pastoris cell contain dna molecular, this dna molecular comprises as at nucleotide acid sequence described in Fig. 1 or dna vector as shown in Figure 4.

Can use any suitable fermentation process in this area in the method.For in the methylotrophy yeast as Pichiapastoris, scale operation is the product on basis by the recombinant DNA of GAP promoters driven, preferably adopts the batch of one three phase, high-cell density to raise fermentation system.In the first phase or vegetative period of this fermentation system, expressive host Pichia pastoris cell is cultivated in minimal medium that limits such as BMGY (buffered limit glycerine complex medium), and wherein non-epigamic carbon source (as glycerine) is excessive.When expressive host Pichiapastoris cell was grown in such carbon source, allogeneic gene expression was suppressed, and made to produce cell mass under the situation that lacks the heterologous protein expression.In this vegetative period, also the pH of preferred culture medium maintains about 5.Next step, expressive host Pichia pastoris cell short period of time in restricted non-inducibility carbon source grows with further increase cell mass, and suppresses glucose and reply promotor.In this limiting growth phase, the pH of substratum remains on below 4, preferably in about 2.0 to about 3.5 scope.Final Issue is production phase, wherein can use " excessive glucose is raised pattern in batches " or " mixed culture is raised pattern in batches ".In " excessive glucose is raised pattern in batches ", add 2% glucose separately." mixed culture is raised pattern in batches ", in fermentor tank, add the non-inducibility carbon source and the glucose of limiting the quantity of, to induce Mo Neilin expression of gene by the GAP promoters driven.

Excretory Mo Neilin chimera protein can reclaim by any methods known in the art from Pichia pastoris substratum.For example, United States Patent (USP) the 3rd, 878, No. 184 and the 3rd, 998, No. 798; Morris and Cagan, Biochim.Biophys.Acta, 1972,261:114-122; Kim etc., protein engineering (Protein Eng.), 1989,2:571-575; With nearest Kondo etc., Nature Biotechnol (Nature Biotechnology), 1997, disclosed method can be used for reclaiming and separating excretory Mo Neilin chimera protein among the 15:453-457.Preferably, expression and excretory chimera protein adopt and comprise that the method for ion exchange chromatography reclaims.More preferably, expression and excretory chimera protein adopt and comprise that the method for CM-Sephadex column chromatography or DEAE-Sephadex column chromatography reclaims.

In another specific embodiment, the invention provides the product of aforesaid method.

6. embodiment

6.1 the preparation of synthetic reorganization Mo Neilin DNA

Proteic aminoacid sequence of reorganization Mo Neilin and the proteic dna nucleotide sequence of coding reorganization Mo Neilin show in Fig. 1.As shown in FIG. 1, the Nucleotide 1-150 1-50 residue of natural Mo Neilin protein B chain of encoding; Nucleotide 150-152 coding is as the glycine that connects " L " part; The encode 1-45 residue of natural Mo Neilin albumin A chain of Nucleotide 153-287.As leading, this aminoacid sequence is corresponding to the signal sequence of methionine(Met) (Met) residue and yeast saccharomyces cerevisiae hybridization pheromone alpha factor: Met-Leu-Leu-Phe-Ile-Asn-Thr-Thr-Ile-Ala-Ser-Ile-Ala-Ala-Lys-Glu-Glu-Gly-Val-Ser-Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala-Glu-Phe (SEQ IDNO:3) by following amino acid sequences for reorganization Mo Neilin albumen.

The synthetic DNA of this coding yeast saccharomyces cerevisiae hybridization pheromone alpha factor signal sequence and reorganization Mo Neilin albumen are by oligomerization M1-M4 and N1-N4 preparation, and these oligomer are the Applied Biosystems 380B dna synthesizer synthetic (seeing Fig. 2-3 and 5) that adopt ACTG company.Oligomer separates with urea one polyacrylamide gel electrophoresis, and through a Sep-pak C18 post (Whatman) purifying, anneals as shown in Figure 3 and is connected, to obtain by EcoRI site sectional synthetic DNA.

Synthetic DNA and reorganization Mo Neilin albumen for composite coding yeast saccharomyces cerevisiae hybridization pheromone alpha factor signal sequence, in 100 μ l PCR reaction volumes, oligomer M2 mixes with oligomer M1 and the N4 of 10pM to each 2pM of N3, under the situation that lacks the Tag archaeal dna polymerase, be heated to 94 ℃ 5 minutes.Reaction mixture slowly is cooled to 37 ℃ then.After adding 1 Vent of unit archaeal dna polymerase (New England Biolabs company), the PCR reaction is carried out according to the method for standard.100 microlitre PCR reaction mixtures contain 50mM Tris-HCl (pH8.0), 2.5mMMgCl ₂, 10mM DTT, 1mM dNTP and 1 Vent of unit archaeal dna polymerase.PCR reaction is carried out as follows: circulate at every turn 94 ℃ 1 minute, 53 ℃ 1.5 minutes, 72 ℃ 2 minutes; 30 circulations altogether.At last, reaction mixture was hatched 10 minutes at 72 ℃.Reaction mixture extracts with phenol/chloroform, uses ethanol sedimentation, with 1.2% low melting-point agarose gel-purified.The dna segment of purifying is inserted into and produces pT7yM plasmid (see figure 5) in pT7bleu (R) carrier (Novagen company).In 20 μ l DNA ligation systems, add 2 μ l 10mMATP, 40 T4 of unit dna ligases (New England Biolabs company), mix with the Mo Neilin dna segment and 50ng pT7blue (R) carrier of 1 μ g purifying.Reaction mixture was 16 ℃ of insulations 16 hours.In 200 μ l intestinal bacteria NovaBlue competent cells, add 5 μ l to connect mixture and will connect mixture and transform into host cell (Messing, Enzymology method, 1983,101:20-78), required sequence is determined (Sanger etc. by adopting T7 and U19 promotor to carry out dideoxy sequence analysis, Proc.Natl.Acad..Sci., 1985.74:5463-5467).

6.2 the preparation of expression vector pGWYS-1

The pGAPZa expression vector is bought from Invitrogen company.Synthetic Mo Neilin dna segment shifts out from pT7yMenallin with EcoRI, and is inserted in the EcoRI site of pGAPZa carrier to obtain pGWYS.In brief, the pT7yMenallin plasmid of 5 μ g purifying digested 2 hours at 37 ℃ with 5 EcoRI of unit (Promega company) in 20 μ l reaction volumes.After reaction mixture separated with 1% low melting-point agarose gel electrophoresis, synthetic Mo Neilin dna segment carried out purifying with Wizard PCR Preps DNA purification kit (Promega company).The Mo Neilin dna segment of 100ng purifying is used for connecting to advance the pGAPZa expression vector.In 10 μ l ligation systems, the pGAPZa carrier of 50ng EcoRI digestion mixes with the Mo Neilin dna segment of 100ng purifying.Ligation exists 10 μ l 20mM Tris-HCl (pH 7.5), 10mMMgCl ₂, 10mM DTT and 200 T4 of unit dna ligases (New England Biolabs company) situation under, spend the night at 16 ℃ and to carry out, to obtain pGWYS.Connecting mixture is transformed in the intestinal bacteria TOP10F ' cell (Invitrogen company).Pick out the clone of 20 anti-Zeocin, insert initiating terminal by PCR reaction examination with alpha factor (5 ' CTATTGCCAGCATTGCTGC3 ') (SEQ ID NO:4) and N4 oligomer.Each clone who selects transfers in 3 milliliters of LB substratum that contain 200 μ g/ml Zeocin, 37 ℃ of vibration overnight incubation.Recombinant plasmid pGWYS adopts Qiagen Tip20 cartridge system (Qiagen company) to prepare from the bacterial cell that 1.5ml cultivates.The pGWYS plasmid of 50ng purifying is used as pcr template to determine to insert initiating terminal.In 25 μ l PCR reaction systems, under the situation that has 1 Taq of unit archaeal dna polymerase (Promega company), 50ng pGWYS plasmid mixes with 2.5pM alpha factor and N4 oligomer.PCR is reflected under the following condition and carries out: each the circulation 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes; 40 circulations altogether.At last, reaction mixture was hatched 10 minutes for 72 ℃.The PCR reaction mixture is after separating on 1.2% sepharose, and one of the clone who contains the inset of required initiating terminal is named as pGWYS-1.The sequence of inset is further determined by dna sequencing.

6.3 transform Pichia pastoris cell with pGWYS-1

For in Pichia pastoris, obtaining Mo Neilin high level and stable expression, the pGWYS-1 plasmid of purifying II type electroporation apparatus (Electroporation Apparatus II) (Invitrogen company), express the described electroporation technology of test kit specification sheets according to Pichia pastoris, transform in the Pichia pastoris cell.In brief, 500ml Pichia pastorisGS115 cell in the YPD substratum 30 ℃ of growths, to OD ₆₀₀Reach 1.3.Cell 4 ℃ through 1, centrifugal 5 minutes of 500g precipitation.The sedimentation cell ice-cold aseptic water washing of 500ml.Repeat rinse step with 250ml and the ice-cold sterilized water of 20ml respectively.Then, cell washes with the ice-cold 1M sorbyl alcohol of 20ml, and is suspended in the ice-cold sorbyl alcohol of 1ml.The yeast GS115 cell 40 μ l in the 1M sorbyl alcohol and the pGWYS-1 plasmid of 10 μ g purifying are mixed to cumulative volume 50 μ l, and mixture is transferred in the into ice-cold cuvette.The cuvette ice bath 5 minutes that contains mixture.Carry out electroporation according to II type electroporation apparatus (production of Invitrogen company) operating parameters.After the electricimpulse, add the ice-cold 1M sorbyl alcohol of 1ml in the test tube, and the content of test tube is transferred in the Eppendorf tube.200 μ l cell transformed place on the 5RDB culture plate that contains 400 μ g/ml Zeocin.Culture plate is hatched until colony at 30 ℃ and is occurred.Positive transfection body is to be grown to feature under the situation about existing at various concentration Zeocin, as 400 μ g/ml, 600 μ g/ml, 800 μ g/ml and 1000 μ g/ml.

6.4 the Detection of Stability of positive transformant

Select three positive transformants,, further determine characteristic according to its growth under the 800 μ g/ml Zeocin situations of existence and the expression of the Mo Neilin that under 2% glucose induction, recombinates.Following experiment is used to detect its genetic stability.Choose each of this 3 positive transformants with aseptic toothpick, do not add on any YPD of being chosen in culture plate 30 ℃ and hatch, occur until colony.Choose these colonies, place on the new YPD culture plate to occur until new colony.After this nonselective growth repeated 50 times, each colony that goes down to posterity was hatched on the selectivity culture plate that contains 800 μ g/ml Zeocin.Protein expression under 2% glucose induction is analyzed with SDS-PAGE.Going down to posterity on the YPD culture plate after 50 times, all three positive transformants show and the same phenotypes of source colony.

6.5 the proteic production of reorganization Mo Neilin

Each three positive transformant selecting in 5.4 is placing 1 liter of YPD substratum of 5 liters of flasks, 30 ℃ of growths, and vibrate strongly (250rpm).After 24,48 and 72 hours, from culturing bottle, obtain the 2ml supernatant liquor respectively.The sample 5 μ l that each time point is collected analyze with 15-20% gradient polyacrylamide gel electrophoresis.It is the protein band of 12kD that excretory reorganization Mo Neilin is observed.The SDS-PAGE quantitative analysis of adopting densometer (Densitometer) (Molecular Dynamic company) to carry out shows that one of positive strain produces the secretion recombinant single chain Mo Neilin near 10 grams per liters.This bacterial strain is named as GWyS1.

6.6 the proteic purifying of excretory reorganization Mo Neilin

Adopt protein method purifying excretory reorganization Mo Neilin from the GwyS-1 yeast strain.According to first method, cultivate after 72 hours, by 12,000rpm (17,000g) centrifugal collection supernatant liquor.After the collection, the pH of supernatant liquor adopts 0.1N NaOH solution to be adjusted to 6.8.1MNaH ₂PO ₄-Na ₂HPO ₄(pH6.8) join in the supernatant liquor with 1: 100 (v/v), and thorough mixing.Then supernatant liquor is loaded on and uses 0.01M NaH ₂PO ₄-Na ₂HPO ₄(pH6.8) on the CM-Sephadex post of solution pre-equilibration (Phamacia company).0.01MNaH with 5 times of column volumes ₂PO ₄-Na ₂HPO ₄(pH6.8) behind the solution flushing post, use 0.3M NaCl-0.01MNaH ₂PO ₄-Na ₂HPO ₄(pH6.8) eluant solution reorganization Mo Neilin.After the water dialysis, proteic purity is defined as about 98% by gel electrophoresis.

According to second method, cultivate after 72 hours, by 12,000rpm (17,000g) centrifugal collection supernatant liquor.After the collection, the pH of supernatant liquor adopts 0.1N NaOH solution to be adjusted to about 7.2.1M NaCl-1M NaH ₂PO ₄-Na ₂HPO ₄(pH7.2) join in the supernatant liquor with 1: 100 (v/v), and thorough mixing.Then supernatant liquor is loaded on and uses 1MNaH ₂PO ₄-Na ₂HPO ₄(pH7.2)-the DEAE-Sephadex post (Phamacia company) of 1M NaCl solution pre-equilibration on.Collect the dialysis of moving phase and water.Proteic purity is defined as about 98% by gel electrophoresis.

Reorganization Mo Neilin albumen according to arbitrary method purifying further is lyophilized into dry powder, to measure its sugariness.

6.7 sugariness and Detection of Stability

The sugariness of the reorganization Mo Neilin of purifying adopts common sense of taste experiment to detect.By carry out the contrast with sucrose sweetness based on the suitable dilution of weight.In the experiment of classics, 1,10,25 and the aqueous sucrose solution of 50mg/ml as standardized solution.Compare the reorganization Mo Neilin albumen of purifying and the minimum weight that sucrose can produce sweet taste.The amount that reorganization Mo Neilin of the present invention need add is approximately lacked 1000 times than sucrose.For example, 50ng/ml reorganization Mo Neilin protein solution is the same sweet with 50mg/ml sucrose (the Lady Lee board sugar in Lucky supermarket).

With 100 μ g/ml concentration, at pH2.0,4.0, dissolve the reorganization Mo Neilin of natural Mo Neilin (Sigma company) and purifying for 6.3 and 7.5 times, to detect stability.Each sample was heated to 37 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃ 15 minutes, and was cooled to room temperature before trial test.The most theatrically be not both, natural Mo Neilin has lost its sugariness when pH2.0 is heated to 50 ℃, even and the reorganization Mo Neilin of purifying still kept its sugariness in 5 minutes 100 ℃ of heating.The present invention is not subjected to limit in this microorganism of describe using or specific embodiment on scope.In fact, from the description of front and relevant chart, this area professional and technical personnel can be very clearly to the various improvement of the present invention except that describing at this.These evolutionary approach belong within the accessory claim scope.

Many documents are quoted at this, and its open part all is incorporated herein by reference.

Claims (49)

1. comprise the isolating nucleic acid of the nucleotide sequence of the chimera protein of encoding, described chimeric protein comprises from the N-terminal to the C-terminal:

A) first peptidyl segment that has at least the aminoacid sequence of 40% identity to form by 1-50 residue with natural Mo Neilin B chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin B chain on determine;

B) peptide base key, or second peptidyl segment forming by 1-12 amino acid; With

C) the 3rd the peptidyl segment that has at least the aminoacid sequence of 40% identity to form by 1-45 residue with natural Mo Neilin A chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin A chain on determine;

Wherein said chimera protein is stable, and than the sucrose of same amount sweet 100 times at least of the described chimeric proteins of given dose, and in described nucleic acid, uses the codon that is preferably used by yeast cell.

2. isolating nucleic acid as claimed in claim 1, wherein said first peptidyl segment is by having at least the aminoacid sequence of 60% identity to form with natural Mo Neilin B chain.

3. isolating nucleic acid as claimed in claim 1, wherein said first peptidyl segment is by having at least the aminoacid sequence of 90% identity to form with natural Mo Neilin B chain.

4. isolating nucleic acid as claimed in claim 1, wherein said first peptidyl segment is made up of the 1-50 amino-acid residue of the natural Mo Neilin B chain of amino-acid residue 1-50 (SEQ ID NO:5) as described in Figure 1.

5. isolating nucleic acid as claimed in claim 1, wherein said second peptidyl segment is made up of amino-acid residue Gly.

6. isolating nucleic acid as claimed in claim 1, wherein said second peptidyl segment is made up of aminoacid sequence Gly-Gly-Gly-Ser (SEQ ID NO:2).

7. isolating nucleic acid as claimed in claim 1, wherein said second peptidyl segment is made up of aminoacid sequence Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser (SEQ IDNO:1).

8. isolating nucleic acid as claimed in claim 1, wherein said the 3rd peptidyl segment is by having at least the aminoacid sequence of 60% identity to form with natural Mo Neilin A chain.

9. isolating nucleic acid as claimed in claim 1, wherein said the 3rd peptidyl segment is by having at least the aminoacid sequence of 90% identity to form with natural Mo Neilin A chain.

10. isolating nucleic acid as claimed in claim 1, wherein said the 3rd peptidyl segment are made up of as the amino-acid residue 52-96 (SEQ ID NO:5) described at Fig. 1 the 1-45 amino-acid residue of natural Mo Neilin A chain.

11. isolating nucleic acid as claimed in claim 1, the amino-acid residue 1-96 of this nucleic acid encoding Fig. 1 (SEQ ID NO:5).

12. isolating nucleic acid as claimed in claim 1, wherein said chimera protein can be by anti-Mo Neilin antibody mediated immunity reaction bonded.

13. isolating nucleic acid as claimed in claim 1, wherein said chimera protein can be by anti-thaumatin antibody mediated immunity reaction bonded.

14. further comprising, isolating nucleic acid as claimed in claim 1, wherein said chimera protein can guide described chimera protein excretory aminoacid sequence from Pichia pastoris.

15. isolating nucleic acid as claimed in claim 14, the endogenous signal sequence that wherein said secretion homing sequence is Pichia pastoris.

16. isolating nucleic acid as claimed in claim 15, wherein said endogenous signal sequence is selected from: Pichia pastoris acid phosphatase, Pichia pastoris aspartate protease and by the signal sequence of the Pichia pastoris carboxypeptidase y of Pichia pastoris PRC1 coding.

17. isolating nucleic acid as claimed in claim 14, wherein said secretion homing sequence is the yeast signal sequence, and wherein said yeast is not Pichia pastoris.

18. isolating nucleic acid as claimed in claim 17, wherein said yeast signal sequence is from the yeast saccharomyces cerevisiae signal sequence.

19. isolating nucleic acid as claimed in claim 18, wherein said yeast saccharomyces cerevisiae signal sequence is selected from: the signal sequence of Saccharomyces Cerevisiae in S UC2 and yeast saccharomyces cerevisiae hybridization pheromone alpha factor.

20. isolating nucleic acid as claimed in claim 19, wherein said yeast saccharomyces cerevisiae signal sequence are the signal sequences of yeast saccharomyces cerevisiae hybridization pheromone alpha factor.

21. isolating nucleic acid as claimed in claim 11 further comprises guiding described chimera protein excretory aminoacid sequence from Pichia pastoris.

22. isolating nucleic acid as claimed in claim 21, wherein said secretion homing sequence are the signal sequences of yeast saccharomyces cerevisiae hybridization pheromone alpha factor.

23. isolating nucleic acid as claimed in claim 14, wherein said secretion homing sequence is selected from: in the Aspergillus giganteus bacterium α-Zhou Qujunsu, alpha-N-Acetylgalactosaminidase, OmpA albumen, mouse α-factor (cCel1), pepper-β-1, and 4-dextranase, the signal sequence of isolating laccase, mouse lysosomal acid alpha-Mannosidase, pig carbonic anhydrase inhibitor, awamori aspergillus tubigensis glucoamylase, the main urine protein of mouse, Pho1, rabbit Zinc metallopeptidase Zace1 (ACE) and the thermally-stabilised α-Dian Fenmei of bacterium from molten wooden fungi Trametes.

24. nucleic acid as claimed in claim 1, wherein said nucleic acid is DNA.

25. comprise isolating nucleic acid with the described nucleotide sequence complementary of claim 1 nucleotide sequence.

26. isolating nucleic acid with the described dna sequence dna hybridization of claim 24.

27. DNA as claimed in claim 24, further comprise can be in Pichia pastoris the pilot protein expression promoter.

28. DNA as claimed in claim 27, wherein said promotor is the endogenous promotor of Pichia pastoris.

29. DNA as claimed in claim 28, wherein said endogenesis promoter are the promotors of Pichiapastoris glyceraldehyde-3-phosphate dehydrogenase.

30. DNA as claimed in claim 24, the amino-acid residue 1-96 of described dna encoding Fig. 1 (SEQ ID NO:5), described DNA further comprise the promotor of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase and the signal sequence of yeast saccharomyces cerevisiae hybridization pheromone alpha factor.

31. DNA as claimed in claim 24 wherein uses the codon that is preferably used by Pichia pastoris cell.

32. comprise the dna molecular of nucleotide sequence shown in Fig. 1.

33. pGWYS1 dna vector as shown in Figure 4.

34. contain the reorganization Pichia pastoris cell that right requires 1 described nucleic acid.

35. contain the reorganization Pichia pastoris cell that right requires 32 described DNA.

36. contain the reorganization Pichia pastoris cell that right requires 33 described DNA.

37. produce the method for chimera protein, this method comprises that cultivation contains the reorganization Pichia pastoris cell that right requires 1 described nucleic acid, makes the chimera protein of encoding by cell expressing and secretion, and reclaims and express and the excretory chimera protein.

38. produce the method for chimera protein, this method comprises that cultivation contains the reorganization Pichia pastoris cell that right requires 32 described DNA, makes the chimera protein of encoding by cell expressing and secretion, and reclaims and express and the excretory chimera protein.

39. produce the method for chimera protein, this method comprises that cultivation contains the reorganization Pichia pastoris cell that right requires 33 described DNA, makes the chimera protein of encoding by cell expressing and secretion, and reclaims and express and the excretory chimera protein.

40. method as claimed in claim 37, wherein said expression and excretory chimera protein reclaim by the method that comprises ion exchange chromatography.

41. method as claimed in claim 40, wherein used ion exchange chromatography are the CM-Sephadex column chromatographies.

42. method as claimed in claim 40, wherein used ion exchange chromatography are the DEAE-Sephadex column chromatographies.

43. the product of the described method of claim 37.

44. the product of the described method of claim 38.

45. the product of the described method of claim 39.

46. the product of the described method of claim 40.

47. the product of the described method of claim 41.

48. the product of the described method of claim 42.

49. a chimera protein, described chimera protein comprises to the C-end from the N-end:

A) first peptidyl segment that has at least the aminoacid sequence of 40% identity to form by 1-50 residue with natural Mo Neilin B chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin B chain on determine;

B) peptide base key, or second peptidyl segment forming by 1-12 amino acid; With

C) the 3rd the peptidyl segment that has at least the aminoacid sequence of 40% identity to form by 1-45 residue with natural Mo Neilin A chain, wherein identity per-cent be with the aminoacid sequence of the identical size of natural Mo Neilin A chain on determine;

Wherein said chimera protein is stable, and than the sucrose of same amount sweet 100 times at least of the described chimeric proteins of given dose, and in described nucleic acid, uses the codon that is preferably used by yeast cell.

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