CN1103374C - Chemical synthesis, expression and recombinant protein production for human serum albumin reformed gene (I) - Google Patents

Abstract

The present invention relates to two fire-new deoxyribonucleic acid sequences for coding human serum albumins, namely a design and manual complete synthesis of a modified gene section of human serum albumins, and a production technology for using methanol yeast engineering bacteria for the large-scale production of gene recombinant human serum albumins. Modified genes can greatly raise the expression quantity of human serum albumins. The production technology can make the structure gene of human serum albumins driven by a methanol inducing promoter obtain high-level expression, and expressed products of the human serum albumins are secreted into a fermentation liquor culture medium. The present invention provides reliable experimental data for the amplification of gene engineering human serum albumins in large-scale pilot production.

Description

Recombinant DNA technology is produced the method for human serum albumin

The present invention relates to use recombinant DNA technology in methanol yeast cell such as pichia pastoris phaff cell the producer gene recombination human serum albumin (Recombinant Human Serum Albumin, HSA).The methanol yeast engineering bacteria in their genome, at least stably be integrated with a copy the coding HSA that imports by experimental technique change structure HSA gene; This changes the control that the structure dna sequence dna is subjected to the high expression level promotor in methanol yeast self source; Under methanol yeast appropriate liquid culture medium culturing condition, the HSA expression product can be secreted in the substratum.The present invention is further relevant is to be fit to design and the chemosynthesis that human serum albumin that methanol yeast expresses changes the structure gene; The structure of expression vector; Methanol yeast transformant, engineering bacteria; Engineering bacteria shakes the genetic expression on the bottle in the laboratory; Carry out high-density high expression level fermentative production rHSA with fermentor tank.

The molecular structure of human serum albumin, gene structure and physico-chemical property thereof

Human serum albumin (HSA) is a kind of protein that contains 585 amino acid whose strand sugar basedization, and molecular weight Mr is 66000-69000 dalton, iso-electric point pI 4.7-4.9.The primary structure of HSA contains 35 halfcystines and a tryptophane Trp214, and wherein 34 halfcystines are combined into 17 pairs of disulfide linkage with covalent; It is oval spherical that the tertiary structure of HSA is, and contains three big protein domains, and each big structural domain includes three minor structure territories, forms with ring-joining region-ring (loop-link-loop) textural association.Before and after 1976, the albuminous amino acid that the Brown professor of Texas ,Usa university and student thereof deliver kinds such as serial paper (comprising 6 pieces of Ph D dissertation) systematic study people, chicken, ox, pig, sheep is formed and protein domain, the amino acid composition of finding the albumin primary structure has high homology and conservative property, and protein is all formed (Peters T (1985) .Adv.Protein Chem.37:161-245 by three structural domains; Brown JR (1976) .Fed.Proc.35 (10): 2141-2144; Meloun B, et al (1975) .FEBS Lett.58:134-137).In recent years, show that with X-ray diffraction crystal structure analysis method the HSA three-dimensional conformation is formed a heart-shaped molecule by three homologous protein domains; Have common motif (motif) in each structural domain; A hydrophobicity pocket (hydrophobic cavity) is arranged between the second and the 3rd structural domain, and this structure participates in many exogenous drug molecules and the transportation of endogenous material in blood circulation.In addition, the topological framework of HSA shows that the HSA main chain backbone contains 67% alpha-helix zone and 16% β-lamella (He XM and Carter DC (1992) .Nature 358:209-215).

HSA is rich in protein in the human plasma, accounts for about 60% of blood plasma total protein.Every liter of human blood contains about 40 grams of HSA, and a normal adult male sex (body weight is in 70 kilograms) has 3.5-4 to rise blood approximately, and 160 gram HSA are promptly arranged in the blood plasma approximately.HSA also is present in juice, skin and the geode of tissue, health except existing in blood plasma, constitutes the outer pond of blood vessel; Reach 350 grams with the outer total HSA of Chi of blood vessel in the blood.Because the permutable total HSA of whole body has 350 grams, human body katabolism every day consumes about 14 grams, and under the normal circumstances, the HSA of katabolism consumption gram number enters blood flow by liver cell synthetic replenishing every day, makes the whole HSA metabolic pool of human body reach running balance.The HSA molecule is 19 days the intravital transformation period the people, mean lifetime 27 days.In these 27 days, the HSA molecule will carry out 15000 times and come and go circulation in the body recycle system, wherein pass through the blood vessel external space (Peters T (1985) .Adv.ProteinChem.37:161-245) 15 times.

Under the Human Physiology condition, HSA not only has the effect of keeping oncotic pressure in the blood (accounting for about 80%), also participates in numerous endogenous material and the transportation of exogenous drug molecule in blood, and the HSA in the blood plays the effect in heavy body material storehouse.Generally the free aglucon of high density that enters blood is by rapidly with after HSA combines, make its free molecule (or ion) density loss in blood, play steady concentration, combine with HSA as aglucon as longer chain fatty acid, metal ion, Triiodothyronine, steroid hormone, testosterone, bilirubin and acetylsalicylic acid, stable equimolecular and form complex body.This species complex arrives the cytolemma near interface of liver, kidney, enteron aisle, brain and other cells of body along blood flow, combine with the HSA membrane receptor on the film, the HSA occurred conformation is changed, impel HSA and aglucon to dissociate, be convenient to these aglucons and utilized by the target area cell, and then the performance biological action.HSA participates in transportation in blood some materials see Table 1.Because HSA is as most important launch vehicle of blood and plasma proteins composition, in body, play a significant role, thereby in clinical medicine practise, has a purposes very widely, be used for treating various shocks in a large number, after burn, war wound, the surgical operation and the various causes of disease such as work accident the hypovolemia patient and chronic nephritis, hepatitis, liver cirrhosis and late malignant tumour patient and malnutritive oedema disease, hypoalbuminemia and the analbuminemia patient etc. that cause.Because the HSA indication is many, consumption big (each patient reaches a few gram scopes with the HSA amount every day), so HSA has the great demand amount.According to document announcement in recent years, at present HSA whole world annual turnover up to 600 tons about, by present 50 dollars/10 grams, annual sales volume is more than 3,000,000,000 dollars.Chinese market is about 70 tons of sales volume in recent years, and annual sales amount is at Renminbi about 3,000,000,000.At present, the demand of HSA in the whole world is the rising attitude

Gesture (Goodey AR (1993) .Trends Biotech.11 (10): 430-433).Up to the present, the HSA product mainly extracts from contribute blood person's blood plasma or people's placenta both at home and abroad.Before and after nineteen forty-six, U.S. Harvard

Some endogenous aglucons that table 1 combines with human serum albumin

Binding constant (KA)Compound M ^-1N calcium ion (divalence) 9 * 10 ²1

8.3 * 10 3 cupric ions (divalence) 1.6 * 10 ¹⁶1 oleic acid (salt) (1) 26.0 * 10 ⁷1

(2) 9.4×10 ⁷ 1

(3) 2.9×10 ⁷ 1

(4) 2.1 * 10 ⁷1 stearic acid (salt) 15.0 * 10 ⁷1 linolic acid (salt) 7.9 * 10 ⁷1 Palmiticacid (salt) 6.2 * 10 ⁷1 lysolecithin 4.3 * 10 ⁴1

Eicosatetraenoic acid 3 * 10 ⁷1

Prostaglandin E1 7 * 10 ⁴2

Lithocholate 9 * 10 ⁷3

Goose dehydrocholate 2 * 10 ⁶3

Cholate 5.5 * 10 ⁴3

Hydrocortisone 5 * 10 ³2

Testosterone 4.2 * 10 ⁴1

Bilirubin 5 * 10 ⁷1

Protoheme 1.1 * 10 ⁸1

L-tryptophane 6.3 * 10 ⁴1

Levothyroxinnatrium 1.6 * 10 ⁶Professor Cohn of 1 university has delivered a piece of writing surplus the serial paper 60 of " blood plasma and protein research ", the processing method (Cohn EJ, et al (1946) the .J Am.Chem.Soc.68:459-475 that from blood plasma, prepare HSA have been systematically discussed in the paper with the cold ethanol precipitator method; Cohn EJ, et al (1947) .J Am.Chem.Soc, 69:1753-1761).At present both at home and abroad the HSA industrialized process for preparing still continue to use the cold ethanol precipitator method and the process modification done slightly (see the Chinese patent publication number, CN1137528A, CN1120439A, CN1042716A, CN1087914A, CN1051913A, CN1123550A).But increasing in recent years people has infected communicable diseases such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C because of input blood product HSA.Owing to reasons such as blood source virus pollutions, moreover country causes HSA in rather short supply to the strict control of blood product import, and people urgently expect to develop new source.Producing recombination human serum albumin Blood substitute source albumin with genetic engineering technique is the most promising in the world present high-tech approach.

The HSA locus is in No. four karyomit(e) of people long-armed (q11-22 of chromosome 4), belong to single copy codominant gene, full length gene 19002bp, in 15 exons are arranged, separated by 14 introns and 13 iteron sequences, form typical eucaryon split gene.The intron of gene is observed the GT-AG rule.Kozak sequence (AXXATG) and cap sequence are arranged in the 5 ' non-translational region of HSA genome type gene; Goldberg-Hogness sequence TATA box (32) as the rna plymerase ii binding site; Enhancer sequence CCAAT box (88) as promoter in eukaryote; Conservative AATAAA sequence is arranged in 3 ' non-translational region, is recognition signal (Dugaiczyk A, et al (1986) the .J Biol.Chem.261 (15): 6747-6757) of 3 ' end polyA tailing.At human hepatocytes, genome HSA genetic transcription goes out the mRNA molecule of 2080 based compositions, a HSA mRNA molecule and 19 rrna constitute a huge polymer, in tenuigenin, carry out the biosynthesizing of HSA, again through Golgi complex processing, modification, sophisticated HSA is secreted into outside the liver plasma membrane, enters blood flow.In Mammals, the genome albumin dna with other three gene linkages of family together, constitute an albumin multigene family, its kinsfolk has: serum albumin, fetoprotein (α-fetoprotein), alpha-albumin (α-Albumin) and vitamin D binding protein (Vitamin D-binding protein, DBP also is GC).Four members of this gene family have the gene structure feature of 15 exons and 14 introns, except the 12nd, 13 2 exons of DBP gene defect, no matter encoded protein matter all exists homology and similarity at the amino acid composition of primary structure or at space structure, show these four family members common ancestral gene (Dugaiczyk A, et al (1996) .J Mol.Biol.259:113-119) that originates from.Investigate from the phylogenetic angle of organism, albumin has been present in batrachians and all more high vertebrates bodies, the small protein molecule (size that is equivalent to three big structural domains of mammalian body words spoken by an actor from offstage protein molecular) that beginning albumin molecule only is made up of 190 amino acid, because animal is more and more evolved to higher organism, neural system, the recycle system, respiratory system, the differentiation gradually of airframe systems such as Digestive tract and urinary system, albumin as the blood plasma major protein, if molecule Xiao Yi drains the needs of physiological functions such as loss from urinary system, make the albumin molecule under the pressure of natural selection progressively from one times of one times of ground of protein (as a motif motif) of a kind of small molecular weight to maximization molecular evolution (Peters T (1985) .Adv.Protein Chem.37:161-245; Putnam FW, TheProteins.2nd ed.Vol.4 Academic Press, London, 1984; Dugaiczyk A, et al (1996) .J Mol.Biol.259:113-119).Human serum albumin gene is the same as whole world different nationalities crowd with another kind of important proteic gene-hemoglobin gene in the human blood, present polymorphism and easy mutagenicity among the race, there is numerous varients in its protein or claims mutant, utilize electrophoretic analysis to identify at present and found 100 various mutations bodies at least, the point mutation that confirms by gene sequencing and the mutant in damaged mutational site have surpassed kind more than 50 (see Table 2 and Fig. 2), wherein detect over half be to find among the Italian crowd, the HSA mutant can cause different physiological roles unusual (Madison J, et al (1994) .Proc.Natl.Acad.Sci.USA91:6476-6480; Arai K, et al (1990) .Proc.Natl.Acad.Sci.USA 87:497-501; Minchiotti L, et al (1987) .Biochim.Biophys.Acta 916:411-418).The HSA mutant is present in and forms alloalbuminemia (alloalbuminemia) in the blood, modal alloalbuminemia is to be present in simultaneously by normal albumin of part and mutant (varient) albumin to constitute so-called bisalbumin emia (bisalbuminemia) in the blood flow

The codon that the type mutant type mutant title of mutant is possible due to the point mutation of table 2 human serum albumin gene changes the change Arg that confirms or infer ^-2→ His Lille, Varese, etc. T/CGT → CAT G → AArg ^-2→ Cys Malmo I, Tradate, etc T/CGT → TGT C → TArg ^-1→ Gln Christchurch, etc. T/CGA → CAA G → AArg ^-1→ Pro Takefu, Honolulu-I T/CGA → CCA G → CArg ^-1→ Leu Jaffna T/CGA → CTA G → TAsp ¹→ Val Iowa City-2, Blenheim A/GAT → GTT A → THis ³→ Gln Nagasaki-3 CAC/A → CAR C → RHis ³→ Tyr Larino CAC/A → TAC C → TGlu ⁶⁰→ Lys Torino T/GAA → AAA G → AAsp ⁶³→ Asn Malmo (no.95) T/GAC → AAC G → AGlu ⁸²→ Lys Vibo Valentia T/GAA → AAA G → AArg ¹¹⁴→ Gly Yanomama-2 C/CGA → GGA C → GGlu ¹¹⁹→ Lys Nagova A/GAG → AAG G → AHis ¹²⁸→ Arg Komagome-2 T/CAT → CGT A → GCys ¹⁷⁷→ Phe Hawkes Bay C/TGC → TTC G → TArg ²¹⁸→ His Honolulu T/CGC → CAC G → ALys ²²⁵→ Gln Tradate-2 C/AAA → CAA A → CLys ²⁴⁰→ Glu Herborn C/AAA → GAA A → GGln ²⁶⁸→ Arg Malmo (no.10) T/CAA → CGA A → GAsp ²⁶⁹→ Gly Nagasaki-1 A/GAT → GGT A → GLys ²⁷⁶→ Asn Caserta G/AAG → AAY G → CLys ³¹³→ Asn Tagliacozzo, etc. T/AAG → AAY G → TAsn ³¹⁸→ Lys Malmo (no.47) AAC/T → AAR C → RAla ³²⁰→ Thr Redhill T/GCT → ACT G → AGlu ³²¹→ Lys Roma T/GAG → AAG G → AGlu ³³³→ Lys Sondrio T/GAA → AAA G → AGlu ³⁵⁴→ Lys Hiroshima-1 T/GAA → AAA G → AGlu ³⁵⁸→ Lys Porto Alegre-1, etc. T/GAG → AAG G → AAsp ³⁶⁵→ His Parklands A/GAT → CAT G → CAsp ³⁶⁵→ Val Iowa City-1 A/GAT → GTT A → TLys ³⁷²→ Glu Naskapi, Mersin, etc. C/AAA → GAA A → GAsp ³⁷⁵→ Asn Nagasaki-2 C/GAT → AAT G → AGlu ³⁷⁶→ Lys Tochigi T/GAA → AAA G → AGlu ³⁷⁶→ Gln Malmo (no.5) T/GAA → CAA G → CGl ³⁸²→ Lys Hiroshima-2 G/GAA → AAA G → AGlu ⁴⁷⁹→ Lys Dublin A/GAA → AAA G → AAsp ⁴⁹⁴→ Asn Casebrook C/GAT → AAT G → AGlu ⁵⁰¹→ Lys Vancouver, etc. A/GAG → AAG G → AGlu ⁵⁰⁵→ Lys Ortonovo T/GAA → AAA G → ALys ⁵³⁶→ Glu Castel di Sangro C/AAG → GAG A → GLys ⁵⁴¹→ Glu Maku A/AAA → GAA A → GAsp ⁵⁵⁰→ Gly Mexico T/GAT → GGT A → GAsp ⁵⁵⁰→ Ala Malmo (no.61) T/GAT → GCT A → CAsp ⁵⁶³→ Asn Fukuoka-1, Paris-2 C/GAT → AAT G → AGlu ⁵⁶⁵→ Lys Osaka-1 G/GAG → AAG G → AGlu ⁵⁷⁰→ Lys B-type (Verona) C/GAG → AAG G → ALys ⁵⁷³→ Glu Milano Fast T/AAA → GAA A → GLys ⁵⁷⁴→ Asn Vanves A/AAA → AAY A → YR=A or G; Y=C or T; Can cause that many human body physiological functions are unusual, as HSA Arg ²¹⁸→ His (CGC → CAC), can cause hyperthyroxinemia (hyperthyroxinemia); The most serious alloalbuminemia type is a kind of analbuminemia (analbuminmia), the patient is often with oedema and hyperlipidaemia hyperlipidemia), its life-span is than agnate crowd's mean lifetime short (Petersen CE, et al (1997) .Biochemistry 36 (23): 7012-7017; Petersen CE, et al (1996) .J Biol.Chem.271 (32): 19110-19117; Dammacco F, etal (1980) .Vox Sang.39:153-161).Human serum albumin gene engineering research present situation

The people such as Lawn of Genentech company in 1981 take the lead in reporting and cloned total length HSA cDNA from people's livers, measured gene order with the terminal termination method of two deoxidations, and then gene codon infers that the HSA mature protein has 585 amino acid, ripe gene front also has 24 the amino acid whose gene orders that comprise of a coding prepro HSA, wherein preHSA is made up of 18 amino acid, is hydrophobicity; Pro-HSA is made up of 6 amino acid, is alkalescence; Its end has the two basic aminoacids processing of the Arg-Arg of Mammals processing that endopeptidase is discerned site.The researchist of Genentech company under the control of intestinal bacteria trp promotor, has obtained to express in the born of the same parents (Lawn RM, et al (1981) .Nucleic Acids Res9 (22): 6103-6114) with encoding mature HSA gene fragment in intestinal bacteria.Nineteen eighty-two, reports such as Nobel laureate professor Gilbert of Harvard University are made probe with mouse albumin cDNA, from people's tire liver cDNA, cloned HSA cDNA by nucleic acid hybridization technique, and in escherichia expression system, having obtained expression, expression product is secreted into cell pericentral siphon (Philipp BW andGilbert W (1982) .J Cell Biochem.Suppl.6:337-346).The HSA of aminoacid sequence that clones' such as Lawn ripe HSA gene is inferred and the previous report of Meloun relatively has 11 amino acid whose differences (Meloun B, et al (1975) .FEBS Lett.58:134-137); With the HSA sequence of Dayhoff report 28 amino acid whose differences (Dayhoff M (1978) .Atlas of Protein Sequence and Structure is arranged relatively, Vol5 (Suppl.3), p266, National Biomedical Research Foundation, Washington); Relatively there is an amino acid different with the HSA aminoacid sequence of generally acknowledging at present, be Glu396 → → Lys (Dugaiczyk A, et al (1986) .J Biol.Chem.261 (15): 6747-6757), thereby think that the HSA that people such as Lawn expresses is a kind of HSA mutant.Nineteen eighty-two, people such as Dugaiczyk have reported with the PCR method and cloned HSA cDNA full-length gene from people's livers, people such as coding HSA mature protein gene and Lawn also have a difference, with the HSA sequence of generally acknowledging at present an amino acid difference is arranged more also, Glu97 → Gly (Dugaiczyk A, et al (1982) .Proc.Natl.Acad.Sci.USA 79:71-75).Aminoacid sequence with the animal of Mammals different genera such as rat, ox and human HSA molecule is made comparisons, and wherein 97 and 396 amino acids residues are Glu (Peters T (1985) .Adv.Protein Chem.37:161-245).From early eighties the nineties up till now, the HSA genetically engineered generally all is the goal gene (see Table 3) of above two HSA sequences as genetic expression.

Use escherichia expression system and express HSA, form the inclusion body form because expression product is present in tenuigenin, its gene engineering product need break bacterium, separate inclusion body, purifying and renaturation; Moreover HSA intramolecule disulfide linkage is many, three-dimensional structure is complicated and the shared volume of the inner atom spatial arrangement of polypeptide chain main chain backbone is big, form the reorganization HSA molecule of folding mistake and the different natural molecule of conformation easily with escherichia coli expression, expression amount is also lower, generally every liter of tens milligrams of levels, thereby be difficult to industrialization (Lawn RM, et al (1981) .Nucl.Acids Res.9 (22): 6103-6114); Latta M, et al (1987) .Bio/Technology 5:1309-1314).With lower eukaryotes yeast expression system expression alien gene, because can be in substratum, and be that well-oxygenated environment helps disulfide linkage and forms in the substratum with the genetic engineered product direct secretion, help giving expression to the protein molecule of native conformation.Express large molecular weight protein hepatitis B surface antigen(HBsAg) (as recombinant vaccine) (the Valenzuela P that succeeds with yeast saccharomyces cerevisiae as nineteen eighty-two, et al (1982) .Nature 298:347-350), thereby with Yeast system expression HSA have an enormous advantage than escherichia expression system.The carrier of yeast expression system-host system divides additional build and integrated two kinds of phraseologies.The recombinant plasmid of additional build is present in the zymic tenuigenin, have in the recombinant plasmid autonomously replicating sequence (autonomously replicating sequence, ARS).Can be in yeast cell self-replicating, and under the promoter element regulation and control expression alien gene, but this recombinant plasmid loses in going down to posterity easily in yeast host cell budding division, pedigree, thereby often is difficult to amplify production with the industrialization large fermentation tank.Integrated expression plasmid is to be incorporated into (single copy or multi-copy integration) in the zymic genome with linearization plasmid or circular plasmids form; duplicate along with yeast host bacterium THE REPLICATION OF CHROMOSOME then; the external source goal gene goes down to posterity in yeast pedigree and tends towards stability; be difficult for losing; the process expressed with the yeast autogene of the process expressed under the regulation and control of Yeast promoter of goal gene is the same in addition; be in nucleus, to transcribe out hnRNA; through modifying and be processed into mRNA; and pass through nuclear membrane and arrive tenuigenin and combine with rrna, express goal gene again.Integrated phraseology is fit to carry out fermentative production (Romanos MA, et al (1992) .Foreign geneexpression in yeast:a review.Yeast 8:423-488) with the industrialization large fermentation tank.Thereby table 3 generally all adopts the integrated phraseology of secretion property with yeast expression system expression HSA.On the other hand, yeast is as the important host bacterium of industrial microorganism, the interior several yeast of a large amount of experiences, especially table 3 aspect industrial fermentation, have been accumulated as manufacture order cell protein engineering strain, existing quite sophisticated production technique aspect the cultivation of large fermentation tank high cell density fermentation.More than the advantage of three aspects lump together, make yeast expression system become the expression system of developing genetically engineered HSA most worthy at present in the world.The research groups such as Okabayashi of the Sleep of Britain Delta biotech company, the Kalman of genetic institute of Hungary academy of sciences and Japanese green cross company

The reference intestinal bacteria Genentech Co USA Nucl.Acids.Res.1981 of table 3 genetically engineered human serum albumin expression system R﹠D institution, 9 (22): 6103-6114 expression system EP0073646 (1983)

Harvard Univ. J Cell Biochem.(1982)

Upjohn，USA EP0079739(1983)

Genetica/Rhone-Poulenc FR2579224(1983)

France Biotechnology (1987) 5 (12): 1309-1314 Bacillus subtilus Genex, USA EP0206733 (1986) expression system EP0229712 (1987)

J Bacteriol (1987) 169 (7): 2917-2925 yeast saccharomyces cerevisiae Genentech Co USA Bio/Technology (1986) 4 (8): 726-730 expression system Delta, UK EP0322094 (1989)

EP0317254(1989)

WO9002808(1990)

Bio/Technology(1991)9：184-187

Rhone-Poulenc，France EP0361991(1990)

Green Cross Co，Japan EP0319641(1989)

EP0399455(1990)

Tonen，Japan EP0366400(1990)

EP0509841(1992)

Tou-Fuel，Japan EP0330451(1989)

JP06090742(1994)

Skandigen/Vepex Nucl.Acids Res.(1990)18(20)：6075-6081

Biotechnika，Hungary EP0308381(1989)

Yeast (1988) 4, S141 schizosaccharomyces pombe Delta, UK WO9001063 (1990) expression system pichia pastoris phaff Phillips Petroleum Pharm.Eng. (1992) 12:48-51 expression system CO, USA P0510693 (1992)

EP0344459(1989)

EP0510678(1992)

Salk Inst. WO9213951(1992)

Biotechnol，USA

Green Cross Co，Japan EP683233(1995)

EP639643(1995)

Res.Corp.Technol, the inferior multiform yeast of USA EP771871 (1997) Chinese Rhein Biotech, Germany Trends Biotechnol. (1992) 10 (12): 413-417 expression system Delta, UK Yeast (1990), S449 transgenic animal Rhone-Poulenc, France WO9303164 (1993) (mouse, rat, rabbit Transgenic.Res. (1994) 3 (6): 365-375 sheep, goat, pig, ox) Genpharm Internayinal, USA WO9108216 (1991)

Inst.Animal Sci， Transgenic Res.(1992)1(15)：195-208

Volcani Center，Israel Transgenic Res.(1994)3(3)：141-151

J Histochem.Cytochem. (1995) 43 (5): the reference kluyveromyces Rhone-Poulenc of 461-470 expression system R﹠D institution, France Yeast (1994) 10:1297-1303 expression system Bio/technol. (1991) 9:968-975

EP0521767(1993)

EP0361991(1990)

Gist-Brocades， US4990447(1991)

Netherlands EP0301670 (1989) transgenic plant Mogen International NV, Biotechnol.NY (1990) 8 (3): 217-221 (potato and tobacco) Leiden, NetherlandsEP: European patent; WO: world patent; US: United States Patent (USP); JP: Japanese Patent is with yeast saccharomyces cerevisiae expression system (Saccharomyces Cerevisiae) secreting, expressing HSA, and every liter of expression amount is (Sleep D, et al (1990) .Bio/Technology 8:42-46 between several milligrams are to 100 milligrams all; KalmanM, et al (1990) .Nucleic Acids Res.18 (20): 6075-6081; Okabayashi K, etal (1991) .J Biochem 110:103-110).Human Kluyveromyces lactis expression system (Kluyveromyces Lactis) secreting, expressing HSA such as the Fleer of France and Blondeau, bottle expression level is shaken every liter of hundreds of milligram level in the laboratory; Made expression rise to the high level (Fleer R, etal (1991) .Bio/Technology 9:968-975) of every liter of 1-2 gram with the high density fermentation technology.Human multiple-shaped nuohan inferior yeast expression system (Hansenula Polymorpha) the secreting, expressing HSA such as goodey of the Hodgkins of Univ Sheffield UK and Delta company express every liter of expression amount with the high density fermentation technological guide and restrain level at 1-2.Dept of Biotechnology of U.S. Philips Petroleum Co. and Sha Ke biotechnology research Sreekrishna and the integrated expression system secreting, expressing HSA (use the high cell density fermentation technology and carry out two-phase fermentation and abduction delivering) of human pichia pastoris phaff such as Barr, every liter of expression has reached the high level (Barr KA, et al (1992) .Protocol for efficient secretionof HSA developed from pichia pastoris.Pharm Eng.12:48-51) of 2-4 gram.Since the nineties, utilizing transgenic plant and animal to produce polypeptide drugs, genetic engineering antibody and recombinant vaccine is an important research direction.The people such as Hoekemu of Holland Mogen company arrive improved cauliflower mosaic virus (Cauliflower mosaic virus with the HSA gene clone, CaMV) downstream of 35S promoter, with recombinant plasmid transformed potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plant, constitute transgenic plant again.For the secretion of exogenous protein, can utilize the prepro sequence (this example preproHSA) of foreign gene self on the one hand; The proteic signal peptide sequence of the PR-S that also can utilize tobacco to secrete outward on the other hand.The result shows that the mosaic gene fragment that PR-S signal peptide sequence and HSA maturation protein constitute can give expression to the correct HSA of processing in the leaf texture of transgenic plant; The transfer-gen plant of clone pre-pro-HSA full-length gene, the guiding peptide can not be processed (Hoekema A, et al (1990) .J Cell Biochem.Suppl.14E, 333 fully by plant; Sijmons PC, etal (1990) .Biotechnology NY 8 (3): 217-221).The people such as Shani of Israel animal science institute utilize sheep beta-lactoglobulin (Beta-lactoglobulin, BLG) promotor, total length HSA gene fragment at downstream clone preproHSA full-length gene cDNA or genome HSA full-length gene and multiple band portion intron sequences, form the transgenic animal (transgenic mice etc.) that contain foreign gene behind the recombinant plasmid transformed sexual cell, express recombinant HSA in the lactational milk of transgenic animal, the transgenic animal expression amount that wherein has reaches 0.3mg/ml (ShaniM, et al (1992) .Transgenic Res.1 (5): 195-208; Barash I, et al (1994) .Transgenic Res.3 (3): 141-151; Barash I, et al (1996) .Nucleic Acids Res.24 (4): 602-610; Hurwitz DR, et al (1994) .Transgenic Res 3 (6): 365-375).

Summary of the invention:

The present invention relates to the design and the synthetic of the DNA sequence (being that HSA changes the structure gene) of two kinds of brand-new coding HSA: according to the HSA aminoacid sequence of Dugaiczyk report and pichia pastoris alcohol oxidase (the alcohol oxidase of Koutz report, AOX1) preference codon of gene, again in conjunction with some other gene design principle integrated design the coding HSA brand-new gene; And then take double-stranded artificial complete synthesis tactful chemosynthesis HSA full-length gene (length is 1849bp) and ripe gene (length is 1779bp), be one of the longest functional gene of chemosynthesis in the world up to now.With the HSA gene transformation pichia pastoris phaff host bacterium GS115 (NRRY-15815) of this chemosynthesis, transformant is through screening, and a kind of pichia pastoris phaff engineering bacteria that increases the HSA expression amount is found in fermentation test.

The present invention relates to a kind of improved expression cassette and be used for production at pichia pastoris phaff reorganization HSA, the structure of expression cassette is as follows:

1) 5 ' control region (including promoter element) in a kind of pichia pastoris phaff bacterium source, this 5 ' control region can be selected alcohol oxidase (AOX1) gene of pichia spp or constructed by dihydroxy acetone synthetase (DAS1) gene etc., control region 3 ' terminal and following 2) sequence be connected;

2) full-length gene of the new gene of above-mentioned coding HSA (promptly utilizing the leader peptide sequence of HSA self to guide goal gene to carry out the secretion expression); With the ripe gene fragment formation mosaic gene (with yeast entogenous leader peptide sequence guide goal gene carry out secretion expression) of S. cervisiae source AMF pre-pro sequence (comprising two codons of yeast KEX-2 proteolytic enzyme processing site Lys-Arg) with the new gene of above-mentioned coding HSA; 3 ' the end and following 3 of these two kinds of HSA structure genes) sequence is connected;

3) 3 ' terminator sequence in a kind of methanol yeast source.

At least be built with in the methanol yeast expression vector among the present invention above-mentioned containing change structure HSA structure gene expression cassette and a kind of for the screening marker gene.This recombinant expression vector transforms methanol yeast host bacterium can use cyclic plasmid form or two kinds of forms of linearizing site-specific integration carrier.The preferential linearization site-specific integration carrier of selecting of the present invention, with in the locus in recombinant expression vector site-directed integration specificity site to methanol yeast (as the pichia pastoris phaff etc.) genome (as being incorporated into AOX1 gene or his4 gene locus), at least stably be integrated with the recombinant vectors that contains above-mentioned expression cassette of a copy with the protoplast transformation method in the engineering bacteria of production HSA involved in the present invention.

In addition, the invention still further relates to a kind of production process: a kind ofly be integrated the engineering bacteria of recombinant vectors that containing of a copy changes the HSA structure gene of structure at least with methanol yeast engineering bacteria scale operation reorganization HSA, shaking under bottle or the fermentor tank high density fermentation condition, HSA structure gene is secreted into the HSA expression product in the substratum under a kind of driving of methanol induction promotor.

The preferential pichia pastoris phaff expression system of selecting of the present invention should be as a kind of modular system of producing HSA with methanol yeast host bacterium, the methanol yeast that other is useful, all can be used as the yeast strain (seeing Table 5) of similar equivalence as many independent bacterial strain, be used to as the host bacterium that produces reorganization HSA from candiyeast, pichia spp, debaryomyces hansenii and four genus of torulopsis.

1981, HSA cDNA was cloned, and also together delivered (Lawn RM, et al (1981) .Nucleic Acids Res 9:6103-6114 according to the determined HSA aminoacid sequence of gene order; Dugaiczyk A, et al (1982) .Proc.Natl.Acad.Sci.USA 79:71-75).1986, people such as Dugaiczyk have cloned HSA genome full length sequence (19002bp) from the human genomic library, there are 15 exons and 14 introns to constitute, and reported by the determined HSA aminoacid sequence of gene order, no matter the latter has corrected the diverse and confused report of in the past some in HSA gene order and aminoacid sequence, be the result who up to the present knows best, the HSA aminoacid sequence of institute of the present invention foundation is exactly report result (Dugaiczyk A, et al (1986) the .Molecular structure of the human albumin gene is revealed by nucleiotidesequence with q 11-22 of chromosome 4.J Biol.Chem.261 (15): 6747-6757) according to this document.

The present invention relates to the design of deoxynucleoside acid sequence of a kind of brand-new coding HSA and manually complete synthesis: according to people's report in 1989 pichia pastoris phaff (pichia pastoris) alcohol oxidase (alcohol oxidase such as the HSA aminoacid sequence of Dugaiczyk report in 1986 and Kouts, AOX1) preference codon (table 4) of gene, brand-new gene (Kouts P, et al (1989) the .Structure comparison of the pichia pastoris alcohol oxidase gene.Yeast 5:167-177 of two coding HSA have been designed again in conjunction with some other gene design principle analysis-by-synthesis; Cregg JM, et al (1989), Functional characterization of thetwo

The codon usage frequency of table 4 pichia pastoris phaff cance high-expression gene AOX1 and AOX2,

Relative synonym usage value and relative tailored index

The relative codon codon of synonym is fit to amino acid code use usage value index number frequency (RSCU) (W) relatively	The relative codon codon of synonym is fit to amino acid code use usage value index number frequency (RSCU) (W) relatively
		Phe UUU 10 0.357 0.217 UUC 46 1.643 1.000 Leu UUA 5 0.309 0.083 UUG 60 3.711 1.000 CUU 17 1.051 0.283 CUC 2 0.125 0.034 CUA 2 0.125 0.034 CUG 11 0.688 0.185 Ile AUU 33 1.500 1.000 AUC 33 1.500 1.000 AUA 0 0.000 0.000 Met AUG 32 1.000 1.000 Val GUU 42 2.333 1.000 GUC 25 1.389 0.595 GUA 3 0.167 0.072 GUG 2 0.111 0.048 Tyr UAU 2 0.066 0.034 UAC 59 1.934 1.000	Ser UCU 38 2.612 0.974 UCC 39 2.690 1.000 UCA 2 0.138 0.051 UCG 2 0.138 0.051 Pro CCU 29 1.333 0.518 CCC 2 0.092 0.036 CCA 56 2.575 1.000 CCG 0 0.000 0.000 Thr ACU 39 1.975 1.000 ACC 33 1.671 0.846 ACA 4 0.203 0.103 ACG 3 0.152 0.077 Ala GCU 50 2.353 1.000 GCC 27 1.271 0.540 GCA 8 0.376 0.160 GCG 0 0.000 0.000 Cys UGU 16 1.455 1.000 UGC 6 0.545 0.375

Ter UAA 1 1.500 1.000 UAG 1 1.500 1.000 His CAU 8 0.348 0.211 CAC 38 1.652 1.000 Gln CAA 21 1.448 1.000 CAG 8 0.522 0.381 Asn AAU 8 0.254 0.145 AAC 55 1.746 1.000 Lys AAA 14 0.368 0.225 AAG 62 1.632 1.000 Asp GAU 21 0.494 0.328 GAC 64 1.506 1.000 Glu GAA 37 0.914 0.842 GAG 44 1.086 1.000

ter UGA 0 0.000 0.000 Trp UGG 18 1.000 1.000 Arg CGU 10 0.923 0.185 CGC 0 0.000 0.000 CGA 0 0.000 0.000 CGG 0 0.000 0.000 Ser AGU 5 0.345 0.128 AGC 1 0.069 0.026 Arg AGA 54 4.986 1.000 AGG 1 0.092 0.018 Gly GGU 89 2.992 1.000 GGC 6 0.202 0.068 GGA 24 0.087 0.270 GGG 0 0.000 0.000

alcohol oxidase genes from yeast，pichia pastoris.Mol.Cell Biol，9，1316-1323)。Pichia pastoris phaff expression system host bacterium wild-type bacterial classification (NRRLY-11430 involved in the present invention, NRRLY-11431, US patent 4,414,329,1983) be a kind of extremely specific thermophilic methanol yeast bacterial strain, under the self-sow state, wherein the alcohol oxidase protein content of alcohol oxidase gene (AOX1) expression accounts for this yeast host bacterium gross protein (being the full gene production spectra-total protein mass spectrum of more than 6000 gene on 1,200 ten thousand base pairs in the pichia pastoris phaff cellular genome) more than 30% (Couderc R and Barratt J (1980) .Oxidation of methanol by the yeast pichia pastoris, purification andproperties of alcohol oxidase.Agricul.Biol.Chem.44:2279-2289).Include the very powerful and strict promoter element (being the AOX1 promotor) that regulated and control by the methyl alcohol substrate in 5 ' the regulation and control zone of the AOX1 gene in this explanation microbial expression system-pichia pastoris phaff expression system host bacteria strain involved in the present invention.The corresponding amino acid whose codon of alcohol oxidase genes encoding shows that by structural analysis being the height codon has a preference for usage (high codon bias usage).This cance high-expression gene only use 25 valid password in 61 coded amino acid codons (effective number of codons, Nc); The frequency that rare codon uses is lower than 10%; (codon bias index, CBI) (codonadaption index, CAI) all up to more than 95%, the alcohol oxidase codon usage frequency sees Table 4 to codon preference index with the codon tailored index.The present invention relates to replace the codon of human serum albumin (HSA) structure gene with alcohol oxidase (AOX1) structure gene preference codon, yet control the gene of the new artificial synthetic coding HSA of being somebody's turn to do of downstream with the AOX1 strong promoter, make the HSA gene in methanol yeast pichia pastoris phaff host bacterium, reach the purpose (sequence and the total synthesis method of strategy, principle and the design of HSA gene brand-new design and the results are shown in embodiment 1,2) of optimum expression.Yet this high expression level HSA engineering bacteria is carried out high-density culture in large fermentation tank, mass production genetically engineered reorganization HSA product (detailed production process is seen embodiment 6).

The present invention relates to replace HSA full-length gene (signal peptide HSA gene self natural signals peptide sequence with alcohol oxidase (AOX1) gene preference codon; Also available yeast host bacterium source signal peptide sequence; The also chimeric signal peptide sequence of available synthetic), insert 5 poly-oligodeoxynucleotides (AAACGATG) in HSA gene A TG front, wherein contain conservative kozak sequence A XXATG (Kozak M (1987) the .Nucleic Acids Res.15:8125-8148 of eukaryotic gene; Kozak M (1990) .Proc.Natl.Acad.Sci.USA 87:8301-8305); AOX1 gene preference codon is replaced the proteic HSA gene fragment of encoding mature, insert the gene order in yeast saccharomyces cerevisiae KEX-2 proteolytic enzyme processing site (Leu-Glu-Lys-Arg) at the gene 5 ' end, again with α-mating factor (alpha-mating factor, AMF) Prepro sequence (85 the amino acid whose gene orders of encode) the formation mosaic gene in yeast saccharomyces cerevisiae source.These two kinds of gene fragments are connected with the AOX1 gene 5 ' control region (pichiapastoris AOX1 5 ' regulation region) of upstream pichia pastoris phaff again, contain promoter element and enhancer element at the AOX1 control region.

Utilize the present invention, the HSA gene obtains the secretion expression in pichia pastoris phaff under the guiding of himself signal peptide sequence or yeast entogenous signal peptide sequence, the expression product direct secretion is in substratum, fermented liquid internal secretion expression levels restrains up to every liter of 5-6, also has the potentiality of further raising expression amount by the optimizing project bacterium of fermentation manufacturing technique.The level of HSA secreting, expressing of the present invention is significantly higher than the level (level of every liter of fermented liquid 1-3.4 gram) of the HSA of methanol yeast (comprising inferior multiform yeast of the Chinese and pichia pastoris phaff expression system) production in the past.

The HSA structure gene that the present invention will be suitable for the synthetic that pichia spp host bacterium expresses is cloned into the centre of the AOX1 5 ' control region and the AOX1 3 ' terminator sequence of pPIC9 expression vector, proofread and correct reading frame, on expression vector, constitute an expression cassette (expression cassette), expression vector (can be cyclic or linearizing) with this gene recombination passes through the protoplast transformation method again, transform the Pichia yeast protoplastis, and then expression cassette stably is incorporated in the genome of host bacterium (being on the karyomit(e)).This expression cassette AOX1 5 ' control region or promotor are used to strengthen the speed that HSA mRNA transcribes; AOX1 3 ' terminator sequence has function and the effect (as impelling hnRNA to add polyA tail etc.) that stops translation of HSA structure gene and stable mRNA transcription product.

Find that in scientific research of the present invention the deoxynucleotide (5-11 gathers the deoxy-oligonucleotide sequence) that inserts the gene 5 ' non-coding region in yeast source before the ATG of goal gene can improve the expression amount of goal gene.The oligodeoxynucleotide that inserts is preferentially selected A and T base.The oligonucleotide sequence that is connected with HSA structure gene ATG is preferentially from AOX1 or constructed by dihydroxy acetone synthetase (dihydroxyacetonesynthase, DAS1) the gene 5 ' end control region sequence of pichia pastoris phaff in the present invention.

Existing several regulating and controlling sequences are identified and are confirmed, can be used for expressing HSA in pichia pastoris phaff.5 ' the control region (promoter element) of the alcohol oxidase of cloning from the pichia pastoris phaff genome (AOX1 and AOX2) gene, constructed by dihydroxy acetone synthetase (DAS1) gene and p40 gene can be used for driving the HSA expression of structural gene in downstream.Preferential 5 ' the control region of selecting of the present invention is for example selected AOX1, (Stroman DW, et al.US Patent 4,855,231) such as AOX2 and DAS1.5 ' the control region that override of the present invention is selected is to select the AOX1 promoter sequence.

In the expression cassette of above-mentioned discussion, also comprise 3 ' terminator sequence.The function of 3 ' terminator sequence is that the structure gene of encoding is played termination, poly-adenosine and stable mRNA.But 3 ' terminator sequence is not considered to very crucial and irreplaceable in the present invention.Cross into middle discovery in scientific research of the present invention, the 3 ' terminator sequence that derives from inferior multiform yeast of the Chinese and pichia pastoris phaff all can be used to the construction expression box.The present invention states those 3 ' terminator sequences from pichia pastoris phaff bacterium source of preferential employing, and for example 3 ' terminator sequence of AOX1 gene, AOX2 gene, DAS1 gene, p40 gene and His4 gene is especially preferentially selected AOX1 3 ' terminator sequence.

The pichia pastoris phaff bacterial classification can be transformed polytype HSA structure gene (as HSA full-length gene and ripe gene).HSA structure gene can be from human liver cell cDNA library, human liver cell cDNA expression library or human genomic library's screening regain (Lawn RM, et al (1981) .Nucleic Acids Res 9:6103-6114; Dugaiczyk A, et.al (1982) .Proc.Natl.Acad.Sci.USA 79:71-75; Dugaiczyk A, et al (1986) .J Biol.Chem.261 (15): 6747-6757); Also can be by the main stream approach thionyl amines legal person worker synthetic HSA gene of present DNA chemosynthesis.The present invention relates to replace HSA structure gene: add the HSA full-length gene by AOX1 gene 5 ' non-translational region 5 poly-deoxy-oligonucleotides with pichia pastoris phaff AOX1 gene preference codon; Yeast saccharomyces cerevisiae source guiding peptide pre-pro-AMF sequence adds two kinds of structure genes of the ripe gene of HSA, sees figure.The HSA structure gene in above-mentioned number of ways source is cloned on a kind of suitable pichia pastoris phaff secretion expression carrier as pPIC9, pPIC9K, pA0804, pA0815, pHIL-S1, pYAM7SP6 etc.The present invention preferentially selects the HSA structure gene and the pPIC9 secretion expression carrier of encoding with pichia pastoris phaff AOX1 gene preference codon.

The plasmid-type carrier is employed as the key element and the support technology of recombinant DNA technology for a long time.Plasmid is a kind of extrachromosomal double-stranded cyclic DNA material that is present in, and it is present in many microbial species.Found that plasmid has the mode of single copy or a plurality of copies to exist in cell.Plasmid vector is as the launch vehicle of pichia pastoris phaff expression system expression alien gene, two kinds of existing waies are arranged in yeast cell: a kind of is to be present in the tenuigenin as cyclic plasmid, this plasmid requires to have self replication sequence (autonomous replication sequence, ARS) exist, being convenient to recombinant plasmid duplicates voluntarily in host's mycetocyte matter, amplification (Cregg JM, 1989, US patent4,837,148), carry out genetic expression under the promoter element of foreign gene in the plasmid expression box drives, Here it is, and there is the outer additional build phraseology of yeast genome in recombinant plasmid.Another kind of plasmid vector is referred to as linearizing site-specific integration carrier (linear site-specific integrative vector), plasmid inside does not have ARS, after recombinant vectors is fabricated and finishes, with a kind of suitable digestion with restriction enzyme linearizing, behind the transformed yeast host bacterium, stably be integrated on the specific site of chromogene group of pichia pastoris phaff with fixing a point.Recombinant plasmid duplicates along with duplicating of yeast chromosomal, is difficult for losing in the yeast division is gone down to posterity; Express under the control of the promotor of foreign gene in expression cassette, this phraseology is referred to as integrated phraseology.The present invention preferentially selects a kind of mode in back, and the HSA structure gene that the present invention soon changes structure stably is integrated in the pichia pastoris phaff host bacterium genome, to reach the purpose of overstable expression.

The present invention has used above-mentioned linearizing site-specific integration type carrier (Cregg JM, 1989, US patent4,882,279; Romannos MA, et al (1995) .Cur.Opin.Biotecnol.6:527-533).These carriers generally have at least following components to constitute: 1) can insert the sequence of dna fragmentation a kind of first time; 2) a kind of alternative marker gene; 3) can insert the sequence of dna fragmentation a kind of second time.A kind of expression cassette that includes foreign structural gene can be inserted between first and second inserted dna fragmentation of this carrier, can at the front or rear face of marker gene.A kind of alternative method is, if 5 ' control region of expression cassette or promoter element are bonded to this inside of inserting one of dna fragmentation, foreign structural gene is connected the downstream of promotor, can insert dna fragmentation with another like this and connect together and can be built into an expression cassette.

This first and second each fragment length that can insert dna fragmentation can not be less than 200bp at least, and the gene order that is integrated the site in nucleotide sequence and the pichia pastoris phaff host bacterium genome has corresponding kinship, as first nucleotide sequence that can insert dna fragmentation and 5 ' the flanking sequence homology that is integrated locus gene, then second can insert dna fragmentation nucleotide sequence should with 3 ' the flanking sequence homology that is integrated locus gene, be convenient to the linearizing recombinant vectors like this and carry out the locus specificity homologous recombination.In addition, several component parts of recombination and integration carrier should directionally be fitted together in order, be configured in the first 3 ' end and second that can insert dna fragmentation as expression cassette and marker gene and can insert 5 ' of dna fragmentation and hold, this kind linearizing recombinant vectors and host bacterium genome are integrated the direction that locus gene inserts after by homologous recombination and also determine like this.Be used for the nucleotide sequence that can insert dna fragmentation as first and second, with the gene order of revising (or displacement) site in the host bacterium natural gene group be homologous.For example, genome is revised (or displacement) site the AOX1 gene locus is taken place, and first and second can insert dna fragmentation and can adopt the AOX1 gene locus, the dna sequence dna of both end sides alar part (comprising AOX1 5 ' control region and 3 ' terminator sequence) so.Can be used as first and second sequences that can insert dna fragmentation in the present invention, select the gene that can in following genome, be separated to: the AOX1 gene that derives from pichia pastoris phaff bacterial strain NRRL Y-11430 and NRRLY-11431, the DAS1 gene, p40 gene and His4 gene (US patent 4,855,231; US patent 4,885,242).

First can insert dna fragmentation inside can comprise controllable control region (promoter element), and it can constitute the control region in the expression cassette.First can to insert dna fragmentation be preferentially to be included in the present invention as the usage of the control region of expression cassette.Fig. 1 is a kind of expression vector synoptic diagram, first can insert dna fragmentation as the 5 ' AOX1 of the control region in the expression cassette in this carrier, (multicloning sites MCS) is configured in the first 3 ' end that can insert dna fragmentation with one 3 ' terminator sequence 3 ' AOX1 for polyclone position.The structure of this linearizing site-specific integration carrier also has an advantage to be to provide a structure gene to insert the site, and need not to add compatible independently a 3 ' terminator sequence again.If first can insert and not contain a kind of 5 ' control region sequence in the dna fragmentation, so a kind of 5 ' suitable control region is inserted into needs at 5 ' end of structure gene, this is for providing a kind of steerable expression cassette necessary, similarly, if second can insert a kind of 3 ' terminator sequence is not provided in the dna fragmentation, so a kind of 3 ' suitable terminator sequence must be connected to 3 ' end of structure gene.This also is that complete expression cassette of formation is necessary.

People are desirably in and include an alternative marker gene on the integrated expression vector of above-mentioned linearizing at least very much.After above-mentioned like this linearizing site-specific integration carrier transformed pichia spp host bacterium, marker gene can be used for the screening and separating from whole transformed bacteria colony of the host bacterium that is integrated with exogenous genetic fragment.Marker gene can be authorized a kind of new phenotypic characteristic of host bacterium that exogenous genetic fragment is arranged by conversion, wild-type host bacterium does not then have, the for example host bacterium recovery that produces a kind of special acid ability, then there is the defective on this special acid biosynthesis pathway in the host bacterium that does not transform foreign gene, perhaps provides certain antibiotic resistance etc.Typical alternative marker gene has from His 4 genes (US patent4,885,242) and Arg 4 genes (US patent 4,818,700) in pichia pastoris phaff and yeast saccharomyces cerevisiae source; Invertase gene (SUC2) (US patent 4,857,467) from the yeast saccharomyces cerevisiae source; Perhaps from the G418/kanamycin resistant gene in intestinal bacteria Tn601 or Tn903 transposon source.In addition, some sequences from bacteria plasmid DNA and phage DNA (as sequences such as Amp, fl ori and ColE1 ori) also are assembled on the expression vector, make recombinant vectors can in bacterial host cell, duplicate, increase, be beneficial to the structure and the evaluation of expression vector.

HSA structure gene and above-mentioned expression vector utilize T behind suitable digestion with restriction enzyme ₄Dna ligase couples together and is built into expression vector, wherein comprises the expression cassette that comprises HSA structure gene of a copy on the expression vector at least.To connect liquid and get the preferential transformed into escherichia coli host bacterium (then transformation efficiency is very low as direct conversion pichia spp host bacterium) in an amount of back, and utilize colibacillary marker gene (as Amp ^r, G418 ^r, and kanamycin etc.), select to contain suitable antibiotic LB culture plate and liquid nutrient medium carries out preliminary screening to transformed bacteria colony, breeding and selective amplification, the extracting bacteria plasmid DNA carries out enzyme and cuts evaluation, filters out the host bacterium that contains HSA structure gene.This host bacterium can prepare a large amount of recombinant plasmids by the plasmid in large scale method for extracting, so that follow-up further evaluation and protoplast transformation.As for further identifying that on recombinant vectors also available multiple technologies are identified: as restriction enzyme evaluation, gel electrophoresis or Southern hybridization technique etc. by the structure of 5 ' control region-expression cassette that HSA structure gene-3 ' terminator sequence is formed and whether in the right direction.

The recombinant plasmid that above-mentioned structure is finished directly or be transformed into pichia pastoris phaff host mycetocyte after linearizing, wherein more sophisticated transformation technology has, as chemical conversion process (Ito, et al (1983) .JBacteriol.153:163-168) with lithium chloride; Electroporation method for transformation (Bio-Rad laboratories 1991, Genepulser transfection apparatus operating instruction and applicationsguide.); Protoplast transformation method (Cregg JM, et al (1985) .Mol.Cell Biol.5:3376-3385).The preferential protoplast transformation method of selecting people such as Cregg to provide in scientific research of the present invention, recombinant plasmid linearizing and identify that recombinant vectors is integrated and use the Southern hybridization technique.

It is methanol yeast that the present invention is used for as genetically engineered host bacterium.Methanol yeast comprises candiyeast (Candida), debaryomyces hansenii (Hansenula), pichia spp (Pichia) and four genus of torulopsis (Torulopsis), and all methanol yeast bacterial strains all can be used as the host bacterium of expression that the present invention changes the HSA structure gene of structure in its table 5.Preferential pichia pastoris phaff and the multiple-shaped nuohan inferior yeast selected in research practice of the present invention, especially preferentially select auxotroph pichia pastoris phaff bacterial strain GS115 (NRRLY-15851), GS190 (NRRLY-18014), PPFI (NRRLY-18017), the Microbiological Characteristics of these bacterial strains is described in detail (US patent 4,818,700 and US patent 4,812,405).Find also that in scientific research of the present invention auxotroph pichia pastoris phaff bacterial strain is convenient to the effect of screening, recombinating and transforming in addition.

5Candida boidinii Hansenula nonfermentansCandida lipolytica Hansenula philodenraCandida mycoderma Hansenula holstiiCandida utilis Pichia farinosaCandida stellatoides Pichia polymorphaCandida robusta Pichia membranaefaciensCandida claussenii Pichia pinusCandida rugosa Pichia pastorisCandida tropicalis Pichia trehalophilaHansenula minuta Torulopsis sonorensisHansenula saturnus Torulopsis candidaHansenula californica Torulopsis bolmiiHansenula mrakii Torulopsis versatilisHansenula silvicola Torulopsis glabrataHansenula polymorpha Torulopsis molishianaHansenula wickerhamii Torulopsis nemodendraHansenula capsulata Torulopsis nitratophilaHansenula glucozyma Torulopsis pinusHansenula henricii Torulopsis bombicol

Wild-type pichia pastoris phaff bacterial strain such as NRRLY-11430 and NRRLY-11431 also can directly change the host bacterium of the HSA structure gene of structure as the present invention, as long as one of clone can be for the marker gene (as SUC2 gene and neo gene etc.) of screening on expression vector, after recombinant vectors transforms the wild-type Pichi strain like this, wherein transformant just can or contain on the antibiotic substratum of G418 at the substratum that contains sucrose and grows, but not transformant then can not grow on this type of substratum, so just can filter out recombinant conversion easily.

After the present invention transforms above-mentioned methyl alcohol host bacterium with linearizing locus specificity recombinant vectors with the protoplastis method, can from host's flora body, screen with the transformant that suitable technology is integrated with conversion HSA expression of structural gene box, as transform mixed solution and be coated on RDHB (the containing the L-Histidine) culture plate, because disappearance group ammonia alcohol dehydrogenase gene (histidinol dehydrogenase gene in the host bacterium genome, his4), thereby can not utilize the L-Histidine, therefore can not grow on the RDHB culture plate forms bacterium colony; And his 4 marker gene are arranged on the linearizing locus specificity recombinant vectors, be integrated into together in the genome of methanol yeast host bacterium in company with HSA expression of structural gene box, can give expression to the Histidine desaturase, thereby growth formation bacterium colony on the RDHB culture plate of L-Histidine can contained, Here it is utilizes microbial nutrition defective type characteristic to screen transformant, on the other hand because site-directed integration, the destruction that causes integration site gene in the host bacterium genome, microorganism also new phenotype may occur, and is slowly still normal etc. as the methyl alcohol utilization.Another kind of commonly used screening method is to utilize neo gene and the zeocin gene gene of marking, add corresponding antibiont (as G418 and kanamycin etc.) in selective medium, recombinant conversion obtains screening owing to growing in containing antibiotic substratum.

Recombinant conversion that above-mentioned screening is obtained is at random behind the some clones of picking, connecing bacterium goes into test tube or shakes bottle and express test, further isolate the cell clone that reaches highest level of expressing HSA, this clone again after increasing as the engineering bacteria prolonged preservation, or directly be used in and use a shake flask fermentation technology or a jar high-density high expression level fermentation technique to carry out in the producer gene reorganization HSA product as the usefulness of planting daughter bacteria, this fermentation technique is seen people's report (Cregg JM such as Cregg, et al (1987) .High-level expression and efficient assembly of hepatitis Bsurface antigenin the methylotrophic yeast, pichia pastoris.Bio/Technology5:479-485; Clare JJ, et al (1991) .High-level expression of tetanus toxinfragment C in pichia pastoris strains containing multiple tandem integrationof the gene.Bio/Technology 9:455-460).

In the present invention, for large-scale industrial production gene recombination HSA product, according to methanol yeast, especially the microbiological property of pichia pastoris phaff-integrated expression system, on fermentor tank, formulated the schedule of operation (fermentation protocol) of the fermentation engineering of three stages, high-density and batch feeding: 1. fs, be also referred to as vegetative period, the engineering bacteria that the highest level that above-mentioned screening and separating is obtained is expressed HSA connects bacterium at a primary yeast basic medium (10 X Basal Salts+250 X PTM as kind of a daughter bacteria ₁+ 8% glycerine) in, the carbon source in this substratum is a kind of non-induction type carbon source (as glycerine).When growing in the substratum that contains this carbon source, this methanol yeast engineering bacterium expression foreign gene is suppressed fully, promptly only allows cell fission, propagation, and cell density increases, and foreign gene is not expressed, and maintains about 5 at the pH of growth phase substratum.2. subordinate phase, this is of short duration period, non-induction type carbon source consumes gradually makes a gift of most induction type carbon source (methyl alcohol) and begins to mend fermentor tank lentamente, cell density in the fermentor tank is in further increase, and the holddown of methyl alcohol reaction promotor (methanol responsivepromoter) is progressively being removed.Progressively adjusted to the pH value of production phase at the pH of this stage substratum, promptly generally at pH5-6, preferentially selecting pH is 5.8.3. the phase III, also claim the production phase, begin to accelerate to mend in the fermentor tank that what the cell density in the fermentor tank had just no longer increases (as mut at this stage induction type carbon source methyl alcohol ^-What bacterial strain), have also has increase slightly (as mut ⁺Bacterial strain), HSA structure gene begins gene expression product to secrete continuously in the fermention medium under methyl alcohol reacts the regulation and control of promotor (as the AOX1 promotor), this fermentation mode is called restriction methyl alcohol batch feeding mode (limited methanol fed-batch mode), and the methanol concentration in whole production stage fermentation jar is limited in the concentration range of 0.2-0.9%.Fermentative production HSA of the present invention, in the phase III, it is the production phase, also can adopt the feed supplement mode (mixed feed fed-batchmode) of mixing, be to mend in the fermentor tank gradually after non-induction type carbon source glycerine adds (2: the 1) mixing by a certain percentage of induction type carbon source methyl alcohol, the characteristics of this fermentation mode are, the cell density in the fermentor tank the production phase also in further increase, the expression cassette that contains HSA structure gene is secreted into HSA in the fermention medium under induction type carbon source methanol induction.Mix the feed supplement mode and especially be fit to do to continuously ferment, continue to produce goal gene product HSA with pichia pastoris phaff host bacterium.

Detail of the present invention will be described in further detail in the following embodiments.

Below general some materials and the method one of all embodiment. bacterial classification:

1. pichia pastoris phaff host bacterium GS115 (his4) NRRLY-15851;

2. e. coli host bacteria:

E.coli Top10F’{proAB，lacI ^q，lacZΔM15，Tn10(Tet ^R)}mcrA，Δ(mrr-hsdRMS-mcrBC)，Φ 80lacZΔM15，ΔLacX74，deoR，recA1，araD139，Δ(ara-leu)7697，galU，galK，epsL(str ^R)，endA1，nupG λ ^-；

E.coli JM109 F’[(recA1，supE44 endA1 hsdR17 gyrA96 relA1 thiΔ(lac-proAB)F’[traD36 proAB ⁺ lacI ^q lacZ ΔM15])；

E.coliHB101(supE44 hsd S20(rB-mB-)recA ₁₃ara-14 proA ₂ lac Y ₁ galK ₂ rpsL ₂₀xyl-5 mtl-1)。

The structure and the preparation of plasmid of above e. coli host bacteria.Two. main agents

1.DNA restriction enzyme, T ₄Dna ligase, polysaccharase etc. respectively available from GIBCO-BRL, Pharmacia,

Bio-Labs and magnificent biotechnology company limited

2. casein hydrolysate (German MERK company product)

3.Bacto-yeast extract (U.S. Difco company product)

4.PCR amplification kit (Sweden Pharmacia company product)

5.DNA sequential analysis test kit (U.S. USB company product)

6. isotropic substance α-P ³²-dATP and γ-P ³²-dATP (Britain Amersham company product)

7. acrylamide (Acr)

N, N-dimethyl bisacrylamide (Bis)

Sodium lauryl sulphate (SDS)

Guanidinium hydrochloride, urea, TEMED (Britain Sigma company product)

8.IPTG, X-gal, DTT, Agarose (Britain Sigma company product)

9.YNB, Biotin, Agar (U.S. Difco company product)

10. sorbyl alcohol, glucose, L-Histidine, L-Methionin, L-methionine(Met), L-leucine, L-are different

Leucine, L-L-glutamic acid (U.S. Sigma company product)

11. glycerine, methyl alcohol (Shanghai chemical reagent factory)

12. enzyme reaction solution

Restriction enzyme high-salt buffer: 10mM Tris-HCl (pH7.5),

100mM NaCl，10mM MgCl ₂

Restriction enzyme medium salt buffer: 50mM Tris-HCl (pH7.5)

50mM NaCl，10mM MgCl ₂

Restriction enzyme low salt buffer: 10mM Tris-HCl (pH8.0)

10mM MgCl ₂

T ₄Dna ligase damping fluid: 50mM Tris-HCl (pH8.0)

10mM MgCl ₂，10mM DTT，1mM ATP

13.SDS-PAGE protein electrophoresis reagent:

Electrophoretic buffer: 192mM glycine, 25mM Tri-HCl, 0.1%SDS, pH8.3

Concentrate the glue damping fluid: 125mM Tris-HCl, 0.1%SDS, pH6.8

Separation gel damping fluid: 375mM Tris-HCl, 0.1%SDS, pH8.8

Sample buffer (1X): 50mM Tris-HCl (pH6.8), 1%SDS, 10% glycerine, 2.5%

Mercaptoethanol, 0.05% tetrabromophenol sulfonphthalein

30% acrylic acid amides: 29% acrylic acid amides, 1%N, N dimethyl bisacrylamide

Coomassie brilliant blue staining liquid: 0.25% (W/V) Xylene Brilliant Cyanine G G-250,5%Hac, 45% ethanol

Destainer: 7.5%HAc, 10% ethanol

14. damping fluid commonly used:

TE damping fluid: 10mM Tris-HCl (pH8.0), 1mM EDTA

STE damping fluid: 10mM Tris-HCl (pH8.0), 1mM EDTA, 20mM NaCl

PBS damping fluid: 10mM NaH ₂PO ₄-Na ₂HPO ₃(pH7.0), 150mM NaCl

10X TBS damping fluid: every liter contains 108 gram Tris alkali, 55 gram boric acid, and 40ml EDTA (0.5M),

pH8.0

50X TAE damping fluid: every liter contains 242 gram Tris alkali, 57.1ml glacial acetic acid, 100ml EDTA

(0.5M)，pH8.0

Saturated phenol: heavily steam the back and use the TE damping fluid saturated, divide loading amount-20 ℃ preservation

Phenol: chloroform: primary isoamyl alcohol (V/V): 1: 1: 0.8

Chloroform: primary isoamyl alcohol (V/V): 24: 1

15. some solution preparations of yeast protoplast transformation method

1.SED:1M sorbyl alcohol, 25mM EDTA, 50mM DTT, pH8.0

2.SCE:9.1g sorbyl alcohol, 1.47g Trisodium Citrate, 0.168gEDTA, pH5.8

3.CaS:1M sorbyl alcohol, 10mM CaCl ₂

4.SOS:1M sorbyl alcohol, 0.3 X YPD, 10mM CaCl ₂

5.CaT：20mM Tris-HCl，pH7.5，20mM CaCl ₂

6.PEG：20％PEG-3350，10mM CaCl ₂，10mM Tris-HCl(pH7.4)

7.1M PBS buffer：132ml 1M K ₂HPO ₄，868ml 1M KH ₂PO ₄，pH6.0。Three. key instrument equipment

1.ABI381A the type automatic dna synthesizer, U.S. Applied Biosystems (ABI) company product;

2. 2.6 liters of fermentor tanks, Japanese MARUBISHI company product;

3. 5 liters of RIBE-5 automatic fermenters, National Engineering Research Center for Biotechnology of East China University of Science product;

4. 50 liters of RIBE-50 automatic fermenters, National Engineering Research Center for Biotechnology of East China University of Science product;

5. 15 liters of Biocenter 15F automatic fermenters are in the biochemical engineering technical study of country of East China University of Science

Heart product;

6. 50 liters of Biocenter 50F automatic fermenters are in the biochemical engineering technical study of country of East China University of Science

Heart product.Three, experimental technique (one) substratum and culture condition

(1%NaCl), solid medium adds Agar powder 15g/L to intestinal bacteria substratum LB for 1%bacto-typtone, 0.5%bacto-yeast extract, and intestinal bacteria are cultivated under 37 ℃ of conditions; Pichia spp rich medium YPD (1%Yeast extract, 2%peptone, 2%dextrose); Pichia spp protoplast regeneration substratum RBD[1M sorbital, 1%dextrose, 1.34%YNB, 4 * 10 ^-5Biotin, 0.005% amino acid mixing liquid (comprising L-L-glutamic acid, L-methionine(Met), L-leucine, L-Isoleucine, L-Methionin)], if solid medium then adds the 2%Agar powder in the liquid medium within; Pichia yeast shake-flask culture base BMGY and BMMY (1%yeast extract, 2%peptone, 100mM PBS buffer, pH 6.0,1.34%YNB, 4X10 ^{- 5}%Biotin, 1%glycerol or 0.5%methanol).Pichia yeast is cultivated under 30 ℃ of conditions.(2) Pichia yeast fermentor tank high density fermentation culture medium and culture condition:

A.10 X Basal Salts：

1.H ₃PO ₄，85％ 42ml

2.CaSO ₄·2H ₂O 1.8g/L

3.K ₂SO ₄ 28.6g/L

4.MgSO ₄·7H ₂O 23.4g/L

5.KOH 6.5g/L

B.250 X PTM1 salts：

1.CuSo ₄·5H ₂O 6g/L

2.KI 0.08g/L

3.MnSO ₄·H ₂O 3g/L

4.Na ₂MoO ₄·2H ₂O 0.2g/L

5.H ₃BO ₃ 0.02g/L

6.CoCl ₂ 0.5g/L

7.ZnCl ₂ 20g/L

8.FeSO ₄·7H ₂O 65g/L

9.Biotin 0.2g/L

10.H ₂SO ₄ 5ml

C.glycerol 8％(V/V)

D.Feed medium 50％glycerol(1L)+12ml/L PTM1

E.Induced medium 100％ methanol+12ml/L PTM1

F.Dissolved O ₂(DO) ＞20％

G.pH 5.0-5.8

The extracting of 30 ℃ of (three) plasmids of H.Temperature

1. plasmid extraction on a small scale

Choosing the mono-clonal bacterium is inoculated in 2ml and contains in the LB substratum of corresponding antibiotic 37 ℃ of shaken overnight.Get 1.5m bacterium liquid next day in the Eppendorf pipe, the centrifugation thalline removes supernatant.Bacterial precipitation is placed ice bath, add successively 100 μ l solution I (50mM Glucose, 25mM Tris-HCl, 10mM EDTA, pH8.0), the vibration mixing, incubated at room 5min; Add 200 μ l solution II (0.2N NaOH, 1%SDS), the upset mixing, ice bath 5min; Add 150 μ l solution III (3M NaAc, pH4.8), the mixing that slightly vibrates, ice bath 5min; Add 450 μ l re-distilled phenol/chloroforms (1: 1), behind the mixing 12, the centrifugal 10min of 000rpm carefully draws the upper strata water in the precooling dehydrated alcohol of 2 times of volumes, placed 2 hours in-20 ℃ of refrigerators, centrifugal 12,000rpm 15min inhales and removes ethanol, carefully add the cleaning of 500 μ l, 70% ethanol again and remove salt, add 18 μ l TE and 2 μ l RNAase enzyme liquid after centrifugal the draining, 37 ℃ are incubated 1 hour, cut for enzyme and identify and further clone and use.

2. plasmid in large scale extracting

Get the bacterium liquid of 500ml overnight incubation, 4 ℃ of centrifugal 10min of following 5000rpm, add 100ml STE, centrifugal again collection thalline after outstanding the washing adds solution I (50mM Glucose successively, 25mM Tris-HCl, 10mM EDTA, pH8.0) 18ml, N,O-Diacetylmuramidase (10mg/ml, 1%SDS) 2ml puts incubated at room 10min; Add solution II (0.2N NaOH, 1%SDS) 40ml, mixing (to bacterium liquid transparent till) gently, ice bath 5min; Add solution III (3M NaAc, pH4.8) 20ml, ice bath 10min behind the mixing again; Add isopyknic phenol: chloroform (1: 1) solution extracting 2 times, dehydrated alcohol-20 a ℃ placement that adds the precooling of 2 times of volumes is spent the night, and 12, the centrifugal 15min of 000rpm, abandon supernatant, precipitation is washed 1-2 time with 70% ethanol, drains the back with 3ml TE dissolving, adds RNA enzyme liquid (10mg/ml) 10 μ l, 37 ℃ of insulation 30min, last Sepharose 2B chromatography column (1 * 10cm uses the TE balance) is collected first peak (macromolecule DNA) with TE (pH7.6) for elutriant.Behind the sample size volume of collecting, the dehydrated alcohol and the 1/10 volume 3M NaAc (pH5.2) that add the precooling of 2 times of volumes, mixing is placed-20 ℃ of refrigerator overnight, 12, the centrifugal 15min of 000rpm abandons supernatant, and precipitation is washed 1-2 time with 70% ethanol, drain back 1ml TE dissolving, get an amount of solution and measure dna content with UV-light spectrophotometer 260nm.Extensive extractive recombinant plasmid dna can be used for the usefulness that protoplasm body of the present invention transforms pichia spp host bacterium and long-term cryopreservation.

(4) conversion of the preparation of competent escherichia coli cell and recombinant plasmid

E. coli host bacteria is inoculated in the 2ml LB nutrient solution 37 ℃ of shaken overnight.Get the 500ml bacterium that spends the night next day and be inoculated in the 50ml LB nutrient solution, 37 ℃ of 300rpm shaking culture 1 hour reach 0.5 OD600 to cell density.The centrifugal 5min of centrifugal 5000rpm under 4 ℃, the supernatant that inclines, thalline add 25ml and contain 100mM CaCl ₂, the cold soln of 10mM Tris-HCl (pH7.4) suspends, and ice bath is placed 30-60min, and then 4 ℃ of centrifugal 5min of 5000rpm, the supernatant that carefully inclines, thalline suspends with calcium chloride cold soln 2ml, behind the placement ice bath 40min, promptly can be used for transforming.

The competence Bacillus coli cells of getting the above-mentioned prepared fresh of 200 μ l is suspended from the Eppendorf pipe, adds an amount of recombinant plasmid dna and connects liquid 10 μ l, ice bath 30min, 42 ℃ of heat-shocked 2min; Add that 500 μ l LB nutrient solutions are slow on 37 ℃ of shaking tables to shake 1 hour with 10, the centrifugal 10sec of 000rpm, most of LB inclines, keep about 200 μ l LB, with the rifle head gently with behind the bacterial suspension mixing, be divided into 50 μ l and 150 μ l, respectively be coated with a LB agarose plate that contains 50 μ g/ml penbritins, cultivated 8-16 hour in 37 ℃ of incubators.

Embodiment 1. is fit to the expression of pichia pastoris phaff host bacterium

The design of human serum albumin full-length gene and ripe gene

The present invention relates to two gene fragments of chemosynthesis HSA full-length gene and ripe gene, its length is respectively 1849bp (609 amino acid of encoding) and 1779bp (585 amino acid of encoding), is one of chemical in the world up to now the longest complete synthesis functional gene.

Design philosophy of the present invention is: utilize at present ideal in the world microbial expression system-pichia pastoris phaff-integrated expression system as the object of the invention expression of gene system; And then utilize powerful promotor alcohol oxidase gene (AOX1) promotor of pichia pastoris phaff host bacterium as the promoter element that drives the object of the invention gene; And then the codon of HSA structure gene replaced with the preference codon of the extreme preferences usage of high expression level of alcohol oxidase gene (AOX1).

The present invention relates to the HSA gene is carried out specifically designing completely newly, adopted molecular biology three big core databases during design: 1. international nucleic acid sequence data storehouse (Gen Bank/EMBL/DDBJ); 2. Switzerland's protein sequence and annotation database (Swiss-PROT); The protein that provides of U.S. Brookhaven National Laboratory and biomolecules three-dimensional structure database (Protein Data Bank, PDB).Adopt multiple computer packages (GENESIS and the PROSIS software package that comprise the Genetic Computer Group of Univ Wisconsin-Madison USA establishment again, the Caltec software package of California Inst Tech USA's establishment, DNASIS that Sweden Pharmacia company provides and PROSIS software package and other program) carry out many-sided assistant analysis, carrying out the HSA gene on computer graphical workstation (SGI R4400) completely newly designs, and consider following principle: the 1.HSA synthetic gene is selected the preference codon of pichia pastoris alcohol oxidase (AOX1) gene preference as far as possible for use, reduce the ratio of the seldom used codon of this gene, wherein this gene preference codon accounts for the HSA synthetic gene codon sum 98% of coding; 2. eliminate the secondary structure (comprising repeating structure, complementary structure, hairpin structure and big segmental reverse palindrome) of the inner complexity that occurs of HSA synthetic gene etc.; 3. eliminated the part restriction endonuclease sites that HSA synthetic gene inside is not suitable for genetic manipulation; Simultaneously inserted Sal I, Hind III, four restriction endonuclease sites of Not I, Xba I successively, made HSA gene relative equilibrium ground be divided into five big fragments, be convenient to splicing, clone and the assembling of synthetic gene in HSA gene inside from 5 ' to 3 ' direction; 3. reduce successive G-C pairing in HSA synthetic gene inside as far as possible, increase the A-T pairing of more pichia spp preference; 4. according to the pichia pastoris phaff expression system, redesigned the reading frame of HSA gene, wherein total length HSA gene is inserted into AOX1 gene 5 ' regulation and control zones (promoter region) with the restriction enzyme BamH I site of 5 ' end, after connect 5 oligodeoxynucleotides of AAACG (including the Kozak sequence of eukaryotic gene), link to each other with the initiator codon ATG of the HSA gene of back again, ripe HSA gene inserts BamH I at 5 ' end, Xho I restriction enzyme site, insert two two basic aminoacids codons of Methionin and arginine after these two restriction enzyme sites, the KEX-2 proteolytic enzyme processing that is used for yeast entogenous is sheared; 3 ' end at the HSA synthetic gene has adopted dual terminator codon TAATAG, strengthens the termination signal of translation, reads over when preventing genetic expression, and end is added an EcoRI restriction enzyme site; 6.HSA full-length gene directly adopts HSA self-priming peptide (comprising 18 amino acid whose signal peptides and 6 amino acid whose leading peptides) sequence to merge in pichia pastoris phaff AOX1 promotor downstream when constituting expression cassette, guides HSA structure gene to carry out the secretion expression; The ripe gene 5 ' end of HSA inserts XhoI restriction enzyme site and two basic aminoacids (encoding sequences Lys-Arg-) of yeast KEX-2 proteolytic enzyme, it is merged mutually with yeast α-mating guiding peptide (85 amino acid are formed) sequence of upstream, this mosaic gene is cloned in pichia pastoris phaff AOX1 promotor downstream, under the driving of AOX1 promotor, carries out the secretion expression of HSA gene.

In sum, the HSA full-length gene and the ripe gene of the present invention's design are seen Fig. 3-4.

Use following formula calculate change structure HSA gene codon preference index (Codon bias index, CBI): $\mathrm{CBI}=\frac{\mathrm{Nopt}-\mathrm{Nran}}{\mathrm{Ntot}-\mathrm{Nran}}$ Wherein: Nopt is the sum that preference codon (Optional Codons) occurs in this gene codon;

Nran is the number that all synonym (Synonymous Codons) are used of equal valuely;

Ntot be this gene codon sum (should with this intragenic coding methionine(Met) (Met),

Except the three class codons such as tryptophane (Trp) and terminator codon).

According to statistics, total length HSA gene, Ntot=600, Nran=9, Nopt=591, $\mathrm{CBI}=\frac{591-9}{600-9}=98.5\%$

Ripe HSA gene, Ntot=578, Nran=8, Nopt=570 $\mathrm{CBI}=\frac{570-8}{578-8}=98.6\%$

Above result shows that the codon preference index of designed total length HSA gene of the present invention and ripe HSA gene reaches 98.5% and 98.6% respectively.

Use following formula calculate change structure gene codon tailored index (codon adaption index, CAI)

CBI＝CAIobs/CAImax (1) $\mathrm{CBIobs}={\left(\underset{k=1}{\overset{L}{\mathrm{\Π}}}\mathrm{RSCUk}\right)}^{1/L}---\left(2\right)$ $\mathrm{CAI}\max ={\left(\underset{K=1}{\overset{L}{\mathrm{\Π}}}\mathrm{RSCUk}\max \right)}^{1/L}---\left(3\right)$ Ground of equal value method of calculation also have: $\mathrm{CAI}={\left(\underset{k=1}{\overset{L}{\mathrm{\Π}}}\mathrm{wk}\right)}^{1/L}---\left(4\right)$ (4) the further abbreviation of formula becomes following formula: $\mathrm{CAI}=\exp \frac{1}{L}\underset{k=1}{\overset{L}{\mathrm{\Σ}}}\ln \mathrm{Wk}---\left(5\right)$ Wherein: the codon tailored index of CAIobs for observing;

CAImax is maximum codon tailored index;

(relative synonymous codon usage RSCU) is relative synonym to RSCU

The usage value;

W is the relative fit value of each codon, and the maximum value of W is 1;

L is that the length of gene (should be with three class passwords such as this intragenic Met, Trp and terminator codons

Except the son).According to statistics, total length HSA gene utilizes following formula to calculate CAI, and correlation values sees Table 4 $\mathrm{CAI}={\left(\frac{\underset{k=1}{\overset{600}{\mathrm{\Π}}}\mathrm{RSCUk}}{\underset{k=1}{\overset{600}{\mathrm{\Π}}}\mathrm{RSCUk}\max }\right)}^{1/600}$ $={\left(\frac{1.632\×2.333\×...\×1.389\×...\×2.992\×3.711}{1.632\×2.333\×...\×2.333\×...\×2.992\×3.711}\right)}^{1/600}$ $={\left(\frac{1.389\×0.368\×0.350\×1.271\×0.357\×0.357\×0.357\×0.357}{2.333\×1.632\×2.353\×2.353\×1.643\×1.643\×1.643\×1.643}\right)}^{1/600}$ $={\left(\frac{3.649\×{10}^{-3}}{153.613}\right)}^{1/600}$

＝(2.404×10 ^-5) ^1/600

=98.2% ripe HSA gene utilizes following formula to calculate CAI, $\mathrm{CAI}={\left(\frac{\underset{k=1}{\overset{578}{\mathrm{\Π}}}\mathrm{RSCUk}}{\underset{k=1}{\overset{578}{\mathrm{\Π}}}\mathrm{RSCUk}\max }\right)}^{1/578}$ $={\left(\frac{1.506\×2.353\×...\×0.368\×...\×2.992\×3.711}{1.506\×2.353\×...\×1.632\×...\×2.992\×3.711}\right)}^{1/578}$ $={\left(\frac{0.368\×0.350\×1.271\×0.357\×0.357\×0.357\×0.357}{1.632\×2.353\×2.353\×1.643\×1.643\×1.643\×1.643}\right)}^{1/578}$ $={\left(\frac{2.659\×{10}^{-3}}{65.844}\right)}^{1/578}$

＝(4.059×10 ^-5) ^1/578

＝98.3％

According to above-mentioned theory calculation result, the codon tailored index of designed total length HSA gene of the present invention and ripe HSA gene reaches 98.2% and 98.3% respectively.

Embodiment 2 is fit to the expression of pichia pastoris phaff host bacterium

The chemosynthesis of human serum albumin full-length gene and ripe gene

The present invention relates to the HSA full-length gene (to call X gene in the following text) and the ripe gene (to call Y gene in the following text) of embodiment 1 design are adopted double-stranded complete synthesis strategy: earlier synthetic complementary oligonucleotide fragment is spliced into 5 big fragments successively according to its sticky end; These 5 big fragments are cloned into respectively on the suitable site of pBluescriptSK carrier, carry out determined dna sequence; These correct 5 big fragments of order-checking further are assembled into the full-length gene and the ripe gene of design.Carry out determined dna sequence again, whether the gene and the design of checking chemosynthesis be in full accord.Concrete chemosynthesis schema following (is example with the X gene).

The chemosynthesis of X gene

Step1. clone Xa, Xb, Xc, Xd and Xe

The chemosynthesis of oligonucleotide fragment

↓

Use the polynueleotide kinase phosphorylation

↓

The annealing of phosphorylation oligonucleotide fragment

↓

Connect with T4DNA ligase

↓

The big fragment of DNA that connects is with suitable restricted

Endonuclease digestion produces sticky end

↓

The big fragment sepharose of gene

Electrophoretic separation and purifying

↓

Big fragment of gene and pBluescript carrier

Connection, transformed into escherichia coli host bacterium

↓

The plasmid preparation is cut evaluation with enzyme on a small scale

↓

Determined dna sequence

(Xa, Xb, Xc, Xd and Xe)

↓

Step2. assemble X gene

(connecting Xa, Xb, Xc, Xd and Xe)

↓

Transformed into escherichia coli host bacterium

↓

The plasmid preparation is cut evaluation with enzyme on a small scale

↓

Determined dna sequence

The assembling Y gene (connects the big fragment of Ya

With the Xb-Xc-Xd-Xe fragment)

↓

Transformed into escherichia coli host bacterium

↓

The small-scale plasmid purchases and enzyme is cut evaluation

↓

Determined dna sequence

X gene and Y gene are divided into oligonucleotide fragment, big fragment particularly and insert suitable restriction enzyme site.Wherein X gene is divided into Xa, Xb, Xc, Xd and 5 big fragments of Xe.Xa further is divided into 20 oligonucleotide fragments, is followed successively by X ₁, X ₂, X ₁₇, X ₁₈, A ₁, A ₂, A ₃, A ₄, A ₅, A ₆, A ₇, A ₈, A ₉, A ₁₀, A ₁₁, A ₁₂, A ₁₃, A ₁₄, A ₁₅And A ₁₆Xb further is divided into 12 oligonucleotide fragments, is followed successively by B ₁, B ₂, B ₃, B ₄, B ₅, B ₆, B ₇, B ₈, B ₉, B ₁₀, B ₁₁And B ₁₂Xc further is divided into 20 oligonucleotide fragments, is followed successively by C ₁, C ₂, C ₃, C ₄, C ₅, C ₆, C ₇, C ₈, C ₉, C ₁₀, C ₁₁, C ₂₁, C ₁₃, C ₁₄, C ₁₅, C ₁₆, C ₁₇, C ₁₈, C ₁₉And C ₂₀Xd is divided into 8 oligonucleotide fragments, is followed successively by D ₁, D ₂, D ₃, D ₄, D ₅, D ₆, D ₇And D ₈Xe is divided into 18 oligonucleotide fragments, is followed successively by: E ₁, E ₂, E ₃, E ₄, E ₅, E ₆, E ₇, E ₈, E ₉, E ₁₀, E ₁₁, E ₁₂, E ₁₃, E ₁₄, E ₁₅, E ₁₆, E ₁₇And E ₁₈The Y gene X gene compares, and only first big fragment Xa is variant, no matter all the other four big fragments and X gene Xb, Xc, Xd and Xe order or dna sequence dna are all identical.Ya further is divided into 18 oligonucleotide fragments, is followed successively by Y ₁, Y ₂, A ₁, A ₂, A ₃, A ₄, A ₅, A ₆, A ₇, A ₈, A ₉, A ₁₀, A ₁₁, A ₁₂, A ₁₃, A ₁₄, A ₁₅And A ₁₆, A wherein ₁To A ₁₆16 oligonucleotide fragments and X gene are identical, see Figure 15.

The chemosynthesis of oligonucleotide fragment and purifying

The above-mentioned oligo DNA nucleotide fragments of all that the present invention relates to synthesizes at the ABI dna synthesizer respectively.After going protecting group, each dna fragmentation uses 16% sex change glue polyacrylamide gel (containing 7M urea) to carry out electrophoresis respectively, after electrophoresis finishes, downcuts suitable band under ultraviolet lamp, with 0.5M NaCl solution soaking, remains on ambient temperature overnight.Use C then ₁₈The post desalination.Elutriant 60% methyl alcohol is collected liquid and is drained with vacuum.The purity of each DNA oligonucleotide fragment is further identified, by γ-P ³²Each the dna fragmentation reaction of dATP isotropic substance end mark is used the 16%PAGE electrophoresis then by polynueleotide kinase (polynucleotide kinase) catalysis, and electrophoresis finishes the back and checks its purity with X-mating plate compressing tablet and radioautograph.The purity of each DNA oligonucleotide fragment that the confession synthetic gene is used should meet or exceed 99%, if purity is not enough, necessary quilt is purifying again.

The preparation of pBluescript SK carrier DNA

X gene and Y gene all are divided into 5 fragments, restriction enzyme site also identical (is example with the X gene):

For big segmental Xa of BamHI-Sal I and Ya, the big segmental Xb of Sal I-Hind III, the big segmental Xc of Hind III-Not I, the big fragment Xd of Not I-Xba I, the big fragment Xe of Xba I-EcoRI, these big fragments are cloned into respectively on the corresponding restriction enzyme site of pBluescript SK carrier.Following scheme (protocol) is a typical method (is example with Xa): 0.05 μ g pBluescript carrier DNA BamHI-SalI double digestion, the carrier DNA of digested mistake Qiagen column purification.Collect the dehydrated alcohol precipitation of liquid with 2 times of volume precoolings, centrifugal 12,000rpm 15min, precipitation is washed 1-2 time with 70% ethanol, abandons ethanolic soln after centrifugal, and precipitating vacuum, to drain freezing preservation standby.

The phosphorylation of oligonucleotide fragment be connected

Be spliced into example with the big fragment of Xa equally, all the other big segmental splicings by that analogy.Each oligo DNA fragment (containing 0.1OD) 10 μ l (the big fragment of Xa has 20 oligo DNA fragments) is added in the 1.5ml Eppendorf pipe, add 100 μ l solution again and (contain 1 μ mole ATP, 20 μ l, 10 X kinase buffer, the polynueleotide kinase of 40 U), mixing is hatched 60min at 37 ℃ gently.75 ℃ of 15min deactivation kinase then add the dehydrated alcohol of 700 μ, 1 precooling, and mixing was placed 2 hours at-20 ℃, and are centrifugal 12, and 000rpm 15min abandons ethanol, and precipitation is washed 1-2 time with 70% ethanol, and is centrifugal, drains, and freezing preservation is standby.

The dna fragmentation of top phosphorylation is dissolved in the 100 μ l TE, and concentration is 1ng/1 μ l.Add oligo DNA fragment mixed solution 20 μ l, add 2.2 μ l 1M NaCl, mixing post-heating to 75 ℃ sex change 2min, annealing gradually in 2 hours then is cooled to room temperature, adds 50 μ l solution (10 X ligase buffer, 7 μ l in this annealing liquid, 1 μ mole ATP, the T of 20 U ₄DNA ligase), 12 ℃ of overnight incubation.

Above-mentioned connection liquid is got the corresponding restriction enzyme enzymolysis of an amount of usefulness, produces corresponding two sticky ends, is convenient to large dna fragment cloning to the corresponding multiple clone site of pBluescript SK carrier (MCS).Can use BamH I-Sal I double digestion as the big fragment of Xa, use phenol/chloroform extracting again, ethanol precipitation reclaims the big fragment of DNA.Get this sample 1 μ l and 2 μ l DNA Marker (100bp ladder) and identify that with the 16%PAGE electrophoresis bromine second dyes, check the size of suitable dna fragmentation.The big fragment of DNA that this kind of enzyme is cut after the evaluation can be used for further clone.DNA large dna fragment cloning after above-mentioned enzyme cut is to pBluescript SK carrier, and (BamH I-Sal I enzyme is cut, 50ng) 10 μ l, 10 X ligase buffer, 4 μ l, the big fragment of Xa DNA (50ng) 10 μ l, T to add pBluescript SK carrier ₄DNA ligase 20 U supply 40 μ l systems with the sterilization distilled water, mixing, and 12 ℃ of connections are spent the night.Get above-mentioned connection liquid 1 μ l and come transformed into escherichia coli HB101 competence host bacterium.Picking 12-18 clone with the double-stranded plasmid DNA of the quick extracting of alkaline denaturation, identifies that with BamH I-Sal I double digestion the result has 3 clones to contain the dna fragmentation of corresponding size at random.Use similar approach, obtained 4 clones' Xb, 7 clones' Xc, 5 clones' Xd, 6 clones' Xe and 9 clones' Ya.

After above-mentioned each positive colony increased, carry out the extracting of double-stranded plasmid DNA in intestinal bacteria, be further purified with Qiagen kit respectively again with plasmid extraction method on a small scale.Make template with the double-stranded DNA behind these purifying, on the full-automatic sequenator of DNA, carry out sequential analysis.The result measures 2 clones' Xa, 2 clones' Xb, and 1 clone's Xc, 1 clone's Xd, 2 clones' Xe and 2 clone Ya, in full accord with the sequence of the gene fragment that designs.

The assembling of X gene and Y gene

The pBluescript SK recombinant vectors that contains Xa, Xb, Xc, Xd, Xe and Ya large fragment DNA that above-mentioned order-checking is correct carries out enzymolysis with BamH I-Sal I, Sal I-Hind III, Hind III-Not I, Not I-Xba I, XbaI-EcoRI respectively.Enzyme is cut product 3% agarose gel electrophoresis respectively, and the adhesive tape with blade downcuts the dna fragmentation that contains corresponding size reclaims dna fragmentation with Qiagen kit again.With 10-50 μ l TE dissolving, make its concentration is 10ng/10 μ l to each large fragment DNA respectively.

Get each 2 μ l (X gene Xa-Xb-Xc-Xd-Xe) of above-mentioned each large fragment DNA, 0.2N NaCl 10 μ l were heated to 75 ℃ of sex change 2min, were annealed to room temperature then gradually at 3 hours.Add 40 μ l solution and (contain 10 X ligasebuffer, 6 μ l, ATP 1 μ mol, T ₄DNA ligase 10 U supply 60 μ l systems with the sterilization distilled water), spend the night 12 ℃ of connections behind the mixing.Get above-mentioned connection liquid 1 μ l and come transformed into escherichia coli HB101 competence host bacterium.36 clones of picking at random with the double-stranded plasmid DNA of the quick extracting of alkaline denaturation, identify with the BamH-EcoRI double digestion, and 10 clones' X gene is screened as a result comes out.Make template with the double-stranded DNA behind the purifying, on the full-automatic sequenator of DNA, carry out sequential analysis.It is in full accord that the result measures the gene order of 2 clones' X gene and design, sees Figure 20-28.

The assembling of Y gene is with the correct X gene of order-checking, with BamHI-Sal I enzymolysis, purified after, will contain the big fragment of Xbcde fragment and Ya and assemble and form.On the full-automatic sequenator of DNA, carry out sequential analysis again.It is in full accord that the result measures the gene of 3 clones' Y gene and design, sees Figure 29-32.

The collection of illustrative plates of X, Y gene restriction enzyme is seen Figure 17-19.

The structure of embodiment 3 expression vector pHSA201 and pHSA301

The full-length gene (1849bp) of chemical synthetic human serum albumin in the embodiment 2 is downcut from pBluescript SK cloning vector with the BamHI-EcoRI double digestion; The proteic HSA gene of encoding mature (1779bp) downcuts from pBluescript SK cloning vector with the XhoI-EcoRI double digestion, fragment is through the 1%Agarose agarose gel electrophoresis, downcut the adhesive tape of corresponding size, reclaim the fragment that kit reclaims goal gene with DNA respectively.Hsa BamHI-EcoRI fragment and HSA XhoI-EcoRI fragment are cloned into respectively on the pichia pastoris phaff expression vector pPIC9 that cuts with corresponding enzyme, the schema that is built into pHSA201 (containing HSA BamHI-EcoRI fragment) and pHSA301 (containing HSA XhoI-EcoRI fragment) recombinant vectors is seen Figure 33-34, and the ligation system is:

HSA dna fragmentation (60ng) 4 μ l T ₄DNA ligase (2U) 1 μ l

PPIC9 carrier (240ng) 5 μ l ddH ₂O 6 μ l

5 X ligase buffer, 4 μ l are in 20 μ l reaction systems, and 16 ℃ of connections are spent the night.

The above-mentioned connection liquid that contains recombinant plasmid pHSA201 or pHSA301 is added 4 μ l and two concentration transformed competence colibacillus of 8 μ l e. coli jm109 host bacterium respectively, coat on LB (the containing penbritin 20 μ g/ml) flat board 37 ℃ of incubator overnight incubation.Then, the conversion bacterium colony that will contain recombinant plasmid pHSA201 or pHSA301 is picking 18 bacterium colonies wherein at random, connect bacterium respectively in 2ml LB (containing penbritin 20 μ g/ml), vibration is 6-8 hour on shaking table, with the quick extracting plasmid of alkaline denaturation double-stranded DNA, cut with BamHI-EcoRI or XhoI-EcoRI enzyme respectively again, the 1%agarose agarose gel electrophoresis, identify the recon that contains the HSA gene that meets corresponding size, wherein identify and contain segmental 4 clones of HSA BamHI-EcoRI, contain segmental 5 clones of HSA XhoI-EcoRI.Connect bacterium in the e. coli host bacteria that contains pHSA201 or pHSA301 recombinant plasmid that comes out from above-mentioned evaluation again, by the extracting of plasmid in large scale extraction process a large amount of contain pHSA201 or pHSA301 recombinant plasmid, be used to transform pichia pastoris phaff host bacterium or prolonged preservation.For the recombinant plasmid application limitations restriction endonuclease Bgl II linearizing that transforms the pichia pastoris phaff bacterium, method is for getting each 20 μ g of recombinant plasmid pHSA201 or pHSA301, with 10X low salt concn damping fluid, add Bgl II restriction enzyme 2 U again, 37 ℃ are incubated 2 hours, make it complete linearizing, again with 75 ℃ of insulations of enzymatic lysis reaction solution 10min, make the deactivation of Bgl II restriction endonuclease, for the usefulness that transforms pichia pastoris phaff host bacterium with the protoplast transformation method.

Embodiment 4. usefulness yeast protoplast transformation methods transform

Recombinant plasmid pHSA201 or pHSA301 are to pichia pastoris phaff host bacterium

Pichia pastoris phaff host bacterium GS115 (NRRLY-15851) is grown on the YPD culture plate, picking mono-clonal GS115 bacterium colony to 5ml YPD liquid nutrient medium, 30 ℃ of shaken overnight.Get 20 μ l bacterium liquid to 100ml YPD liquid nutrient medium, 30 ℃ of shaken overnight.100ml bacterium liquid at the room temperature centrifugal 10min of 1500g, is abandoned supernatant; With 20ml aqua sterilisa suspension yeast cell, with the centrifugal 10min of 1500g, abandon supernatant in room temperature; With 10ml SED suspension yeast cell, with the centrifugal 5min of 1500g, abandon supernatant in room temperature; With 10ml sorbyl alcohol suspension yeast cell, with the centrifugal 5min of 1500g, abandon supernatant in room temperature; With 10ml SCE suspension yeast cell, (helicase, Sigma company product 3mg/ml), are hatched 30-45min at 30 ℃, and it is good making yeast host bacterium about about 70% form protoplastis to add 3 μ lZymolgase; With the yeast protoplastis centrifugal 10min of 750g, abandon supernatant in room temperature; Add 5ml 1M sorbyl alcohol suspension yeast protoplastis, with the centrifugal 10min of 750g, abandon supernatant in room temperature; Add 5ml CaS suspension yeast protoplastis, with the centrifugal 10min of 750g, abandon supernatant, add 0.6-1ml CaS suspension yeast protoplastis (in 30min, using) again for transforming in room temperature.

Getting above-mentioned pichia pastoris phaff bacterium protoplasma body fluid 100 μ l adds respectively among the embodiment 3 with the linearizing recombinant plasmid pHSA201 of BgI II restriction enzyme, pHSA301 or the unloaded carrier of pPIC9 (as the usefulness of the contrast of destination gene expression), each 10 μ l (containing 5 μ g linearizing dna fragmentations) is at incubated at room 10min; Every pipe adds 1ml fresh preparation PEG/CaT (1: 1) solution, incubated at room 10min behind the mixing; With the centrifugal 10min of 750g, abandon supernatant in room temperature, blot all liquid in the Eppendorf pipe with thieving paper; In every pipe, add 150 μ l SOS solution again, incubated at room 20min, in every pipe, add 850 μ l Sorbitol Solution USPs again, fully behind the mixing, getting 100-300 μ l conversion fluid again in every pipe adds to 10ml and dissolves the back insulation in 45 ℃ RD, behind the mixing, be laid on the RDB plate fast, in 30 ℃ of incubators, be inverted and cultivated 4-6 days.On the RDB culture plate, can grow many monoclonal positive bacterium colonies of recombinant plasmid that contain.

Embodiment 5. human serum albumin change the expression of structure gene in pichia pastoris phaff

With the recombinant vectors pHSA201 that contains linearizing site-specific integration carrier that transforms with the protoplast transformation method among the embodiment 4, the positive transformant of the unloaded vector expression box of pHSA301 or pPIC9 is distinguished 24 clones of picking at random from the RDB culture plate, be inoculated in the 4ml GMMY nutrient solution, 30 ℃ of shaking table joltings 36-48 hour, the centrifugal 10min of 5000rpm, respectively get 100 μ L supernatant liquors, vacuum is drained, sample is carried out the 6%SDS-PAGE electrophoresis respectively to be identified, compare with the unloaded vector expression thing of pPIC9, identify high-expression clone, 4 clones that filter out that wherein contain the pHSA201 recombinant plasmid contain 3 clones that filter out of pHSA301 recombinant plasmid.The high-expression clone that these are screened is protected bacterium, slant culture with 15% glycerine low temperature respectively then, is used for as the engineering bacteria of further expressing or does Southern hybridization and methyl alcohol utilizes the usefulness of evaluation such as phenotype.

The colony inoculation of the high-expression clone of the above-mentioned through engineering approaches of picking (containing pHSA201 or pHSA301 recombinant plasmid) in 4ml GMGY liquid nutrient medium, 30 ℃ of shaking table shaken overnight.Getting the 1ml yeast liquid transfers in 50ml BMGY nutrient solution, continue jolting 18-24 hour, connecing bacterium amount by 4% again connects 40ml kind daughter bacteria and goes in the 1000ml GMGY substratum, continue vibration 24-30 hour at 30 ℃ of shaking tables, nutrient solution with the centrifugal 5min of 5000rpm, is abandoned supernatant, and these two kinds of engineering bacterias are used 200ml BMMY abduction delivering substratum suspension thalline respectively then, be placed on 30 ℃ of shaking tables and continue vibration 3-4 days, added (adding to final concentration with 100% methyl alcohol is 0.5%) every 24 hours.At this moment the cell density of pichia pastoris phaff engineering bacteria has generally reached 18-20 OD ₆₀₀, the foreign protein great majority of genetic expression are secreted in the liquid nutrient medium.Fermention medium is at 4 ℃ of centrifugal 20min of 10000rpm, abandon thalline, the supernatant liquor that contains a large amount of HSA expression products, available cold ethanol (alcohol concn 6%) precipitation, Sephadex G-25 and Sephadex G-75 gel exclusion chromatography purifying, again further on FPLC MONO Q HR 5/5 and Superdexp30 chromatography column be further purified, the protein peak effluent liquid that column chromatography is collected is pumped into dry powder with freeze drier, sample can be used for the SDS-PAGE electrophoresis identifies that Western-blot analyzes, n terminal amino acid sequential analysis and peptide mapping, do the analytical work of reorganization such as HSA comformation in solution evaluation HSA.

The high density fermentation test of embodiment 6. pichia pastoris phaff engineering bacterias on the 50L fermentor tank

The scale operation of one recombination human serum albumin

In the present embodiment the pichia pastoris phaff engineering bacteria is carried out the high-cell-density cultivation fermentation test with feed supplement-batch fermentation mode (Fed-bach-fermentation) respectively on 2.6L, 5L, 15L and 50L fermentor tank, purpose is to explore the sero-abluminous approach of scale operation gene recombinant human.

Pichia pastoris phaff engineering bacterial strain GS115:pHSA201 or GS115:pHSA301 are grown on the YPD flat board, and picking mono-clonal colony inoculation is in the BMGY liquid nutrient medium, and vibration is 24 hours on shaking table.To plant daughter bacteria and transfer in 250ml BMGY liquid nutrient medium, on shaking table, continue vibration 24 hours.To plant daughter bacteria again transfers in the basic medium in the 5L fermentor tank.Basic medium is by 10 X Basal salt+250 X PTM ₁+ 5% (w/v) glycerine+0.05% bubble enemy forms.Plant daughter bacteria and cultivate 20-24h in the 5L fermentor tank, cell density reaches 130-150 OD ₆₀₀, it is connect bacterium to the 50L fermentor tank.The 50L fermentor tank holds basic medium 25L, and basic medium is by 10 X Basal salt+250 X PTM ₁+ 8% (w/v) glycerine+0.05% bubble enemy forms.Growth conditions comprises in batches: pH=5.5-5.8 (by the control of 25% solution of ammonium hydroxide); Leavening temperature T is 30 ℃; Dissolved oxygen amount (DO) in the fermentor tank is greater than 20% air saturation.

Methanol yeast engineering bacteria growth in the fermentor tank about 24 hours, the glycerine as carbon source in the basic medium is exhausted basically fully.A kind of glycerine feed supplement of restriction begins to import in the fermentor tank by peristaltic pump then, and feed supplement liquid includes 50% (w/v) glycerine+12ml/L PTM ₁, the feed supplement time is 17-28 hour, feed supplement speed is 200-260ml/ hour.After restrictive glycerine feed supplement finished, a kind of methanol feeding slowly began to carry out, and feed supplement liquid includes 100% methyl alcohol+12ml/L PTM ₁, feed supplement speed only is 10-15ml/ hour during beginning, through the slow feed supplement of several hrs, engineering bacteria occurs the culture reaction of methyl alcohol restriction in jar.This culture reaction shows as the minibreak of dissolved oxygen amount increase suddenly and methanol feeding.Afterwards, methanol feeding speed begins to accelerate, the methanol concentration in jar after this whole abduction delivering period by online detection, feed supplement speed is automatically adjusted by the computer programming process control, keeps the interior methanol concentration of jar interior substratum between 0.2-0.9%.In the different time points of whole fermentation process, the total protein in the fermented liquid is carried out on-line tracing detect: the empirical equation that the typical curve of being done with the blood product human serum albumin is tried to achieve, calculate the interior total protein content of fermented liquid.

Expression amount to secreting, expressing HSA in every batch fermentation liquid in the fermentor tank carries out quantitative analysis.It is some to get every batch fermentation liquid, carry out centrifugal 5000g * 20min, abandon thalline, supernatant liquor through concentrate, after the G-25 desalination, sample carries out the SDS-PAGE electrophoresis, dyes with Xylene Brilliant Cyanine G then, again electrophoretic band optical density method is carried out quantitative analysis, and make standard with the area of the SDS-PAGE electrophoresis-Xylene Brilliant Cyanine G electrophoretic band of the different concns of natural blood product HSA pure samples, and to carry out the expression amount of the reorganization HSA that produces in the fermentor tank is done quantitative analysis, some fermentation indexs see Table 6.

Table 6 high density fermentation technology producer gene recombination human serum albumin batch bacterial strain pH methanol induction time dry cell weight HSA expression amount

(hour) (g/L) (g/L) 1 GS115:pHSA201S ₁5.78 101 79 2.65 2 GS115: pHSA201S ₂5,85 168 185 5.68 3 GS115:pHSA201S ₃5.80 192 163 5.96 4 GS115:pHSA201S ₄5.90 258 169 4.67 5 GS115:pHSA301S ₁5.76 102 80 2.45 6 GS115:pHSA301S ₂5.85 168 178 5.53 7 GS115:pHSA301S ₃5.80 below 256 152 4.58 is description of drawings, wherein:

Fig. 1 makes HSA gene relative equilibrium ground be divided into five big fragments for a kind of expression vector synoptic diagram Fig. 2 inserts Sal I, Hind III, four restriction endonuclease sites of Not I, Xba I for HSA full-length gene and the ripe gene map 5 that the present invention designs in HSA gene inside from 5 ' to 3 ' direction for HSA full-length gene and the ripe gene map 4 that the present invention designs by the point mutation of gene sequencing confirmation and the mutant Fig. 3 in damaged mutational site successively.Xa in Fig. 6 aX gene, Xc in Xb gene order Fig. 6 bX gene, Xd gene order and Xe portion gene sequence chart 6c Fig. 6 a, the continuous figure of 6b, Xe portion gene sequence in the X gene, connect in 6b Fig. 7 aY gene Ya, Xc in Xb gene order Fig. 7 bY gene, Xd gene order and Xe portion gene sequence chart 7c Fig. 7 a, the continuous figure of 7b, Xe portion gene sequence in the Y gene, the schema Figure 27 of schema Figure 26 pHSA301 recombinant vectors of sequential analysis Figure 25 pHSA201 recombinant vectors that connects the sequential analysis Figure 21-24 clone Y gene of 6b Fig. 8 a-gX gene and Y gene oligonucleotide fragment Fig. 9 X gene restriction map spectrogram 10Y gene restriction map spectrogram 11-12X and the Y gene restriction map spectrogram 13-20 clone X gene N-end of HAS behind protein separation of recombinating analyzed

Claims (19)

1, recombinant DNA technology is produced the method for human serum albumin, comprise cultivating and contain the methanol yeast host bacterium that the coding human serum albumin changes the structure expression carrier, and therefrom reclaim human serum albumin, it is characterized in that: described coding human serum albumin changes the nucleotide sequence that the structure gene comprises the 84-1838 position shown in Fig. 3 a-3c.

2, according to the process of claim 1 wherein that the coding human serum albumin changes the structure gene and comprises 1) nucleotide sequence of the 12-1838 position shown in Fig. 3 a-3c, or 2) nucleotide sequence of the 12-2021 position shown in Fig. 4 a-4c.

3, according to the method for claim 2, wherein before human serum albumin leader peptide sequence ATG, insert 5 poly-oligodeoxynucleotide AAACGATG, wherein contain the conservative Kozak sequence A XXXATG of eukaryotic gene.

4, according to the process of claim 1 wherein that expression vector comprises:

1) 5 ' control region in a kind of pichia pastoris phaff bacterium source, include promoter element, this 5 ' control region is selected from alcohol oxidase AOX1 gene, constructed by dihydroxy acetone synthetase DAS1 gene or the histidinol dehydrogenase HIS4 gene in pichia spp source, control region 3 ' terminal and following 2) sequence be connected;

2) nucleotide sequence of the nucleotide sequence of the 12-1838 position shown in Fig. 3 a-3c or the 12-2021 position shown in Fig. 4 a-4c;

3) 3 ' terminator sequence in a kind of methanol yeast source, this 3 ' terminator sequence is selected from 3 ' terminator sequence of AOX1 gene, AOX2 gene, p40 gene or the His4 gene in pichia pastoris phaff source.

5, according to the method for claim 4, wherein expression vector also comprise at least one can be for the marker gene of screening and the dna fragmentation of a reproducible replication orgin in e. coli host bacteria.

6, according to the method for claim 4, wherein expression vector is a cyclic plasmid.

7, according to the method for claim 4, wherein expression vector contains the full-length gene of the coding human serum albumin of a copy at least.

8, according to the method for claim 4, wherein the promoter element of expression vector and 3 ' terminator sequence all are the dna fragmentations that obtains from pichia pastoris phaff AOX1 gene isolation.

9, according to the method for claim 4, wherein expression vector contain multiple copied 1)-3) and sequence.

10, the mode that is connected with head-tail between the sequence according to the method for claim 9, wherein 1 of multiple copied)-3) is cascaded.

11, according to the method for claim 4, wherein expression vector is pHSA201 shown in Figure 25.

12, according to the method for claim 4, wherein expression vector is pHSA301 shown in Figure 26.

13, according to the method for claim 4, wherein expression vector is the linearized vector behind the restriction enzyme Bg1 II enzyme.

14, according to the method for claim 4, wherein methanol yeast host bacterium is selected from the methanol yeast of pichia pastoris phaff host bacterium GS115NRLLY-15851 or candiyeast, debaryomyces hansenii and three genus of torulopsis.

15, according to the method for claim 4, wherein to cultivate methanol yeast host bacterium and shake a bottle scale acquisition high level expression at 1 liter, the time of methanol induction is 4-8 days.

16, according to the method for claim 4, wherein every liter of methanol yeast host bacterium in the 100-185 gram that weighs.

17, according to the method for claim 4, wherein every liter of expression of human serum albumin gene restrains up to 4-6.

18, according to the method for claim 4, wherein the methanol yeast host bacterium of Pei Yanging has Mut ^-Phenotype.

19, according to the method for claim 4, wherein the methanol yeast host bacterium of Pei Yanging has Mut ⁺Phenotype.

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