CN1283699A - Multi-expression kit for multiple promoters contained in methanol yeast expression system - Google Patents

Abstract

The present invention is an improvement on methanol yeast expression system to provide multi-expression kit of more promotors contained in mothanol yeast expression system. By searching the promotors without conflict in activation expression, the expression kits of foreign genes are respectively constructed. After they are integrated in a single yeast cell, the gene engineering strain containing more expression kits of more promotors is obtained. By inducing or selecting proper promotor, the growth and expression can be conducted as same time to shorten fermenting time and simplify fermenting condition.

Description

The many expression cassettes of multiple promotor that contain in the methanol yeast expression system

The present invention relates to the methanol yeast eukaryotic expression system, especially contain the many expression cassettes of multiple promotor and in the methanol yeast eukaryotic expression system, efficiently express foreign protein about application.

Methanol yeast Pichia pastoris expression system is the eukaryotic expression system that Invitrogen company successfully develops.Because characteristics such as but its expression amount height secreting, expressing and foreign protein be few, existing in recent years a large amount of foreign proteins successfully obtain expressing (Ohtani W, Ohda T, Sumi Aet al.Anal Chem 70:425～429,1998) in the methanol yeast system.Generally comprise in the methanol yeast expression plasmid: promotor that exogenous protein expression promotor, multiple clone site, 3 ' terminal transcription termination sequence (AOX1 3 ' end sequence) and expression resistant gene are required and replicon etc.For secretion character grain, between promotor and multiple clone site, also there is the secretion signal peptide sequence.In the multiple clone site behind foreign gene insertion promotor or the signal peptide.Promoter sequence in the expression plasmid or 3 ' end sequence are by being integrated in the yeast chromosomal exogenous gene expression box with Pichia pastoris chromosomal DNA generation homologous recombination, by the expression of corresponding method induction exogenous gene with promotor, so the selection of promotor has very important influence to the expression of foreign protein.

Can be used for the methanol yeast expression promoter and derive from methanol yeast itself mostly, as pAOX (pAOX1 and pAOX2), pDAS, pP40, pGAP and pFLD etc.Different promotors has different activated transcription characteristics according to the effect of its natural albumen of transcribing in yeast is different, and it also has nothing in common with each other by force.According to the existing report of Pichia pastoris expression system, wherein alcohol oxidase promotor pAOX1 is to use at most, expression efficiency is very high one.PAOX1 is a very strong promotor,, shakes in the bottle and just can obtain very high expression amount through the inducing of methyl alcohol with the exogenous protein expression engineering bacteria of its structure.PGAP and pFLD promotor then are (Shen-S, the et al.Gene216 (1): 93-102,1998 that just is in the news recently; Doring-F, et al.Biochem Biophys Res Commun, 250 (2): 531-535,1998), according to people such as Doring, in Pichia pastoris expression system, the pGAP promotor is more suitable for having in expression the Mammals translocator of function than pAOX1.In addition, derive from Candida, the eukaryotic promoter of Hansenula and Torulopsis also can be used for the exogenous gene expression of methanol yeast.

Yet existing patent and pertinent literature show that (US 5643792; H.R.Waterham, et al Gene 186 (1): 37-44,1997), the pAOX1 expression system that is used in a large number now is owing to the toxicity of methyl alcohol pair cell and the glycerine reasons such as weak inhibition to methanol induction, must be earlier in fermentor tank through glycerine vegetative period of 1-2 days, then limit the quantity of and add glycerine and make cell grow into proper density, just add methyl alcohol at last gradually and begin abduction delivering, the whole cycle reaches 7-12 days, and the very difficult control of the method for this abduction delivering, so the high efficiency of pAOX1 promotor often can't show fully.Though and promotors such as pGAP can the constitutive expression foreign protein, for most foreign proteins, adopt its expression amount and unsatisfactory after the pGAP promotor, this causes it to use that also to can not show a candle to pAOX1 extensive.

As the most successful example of methanol yeast expression system be in methanol yeast Pichia pastoris, efficiently express human serum albumin (Human Serum Albumin, HSA).HSA is the great important clinical medicine of a demand.Because spreading of illness such as acquired immune deficiency syndrome (AIDS) and hepatitis makes the HSA medicine in blood source have the threat of carrying virus, and the time report of relevant accident arranged.Therefore exploring the research and development of genetically engineered HSA medicine over 15 years in the world, wherein methanol yeast Pichia pastoris expression system is the most successful one always.The be expressed as external secretion type of HSA in methanol yeast, the promotor of employing is pAOX1, it shakes a bottle expression amount and is about 50mg/l.Xiang Guan patent has US5707828 therewith, EP 0570916A2, EP 0510693A2 etc.Therefore, set up and newly in the methanol yeast contain multiple many expression cassettes of promotor constructing system, make and adopt more simply and easily that fermentation condition can obtain efficiently expressing of foreign protein, have important use and be worth.

The objective of the invention is to existing methanol yeast Pichia pastoris expression system is improved, provide the multiple promotor that contains in the methanol yeast expression system many expression cassettes, make it possible to adopt more simply and easily that fermentation condition can obtain efficiently expressing of foreign protein, reduce industrial cost on the whole.

The invention provides the many expression cassettes of multiple promotor that contain in a kind of methanol yeast expression system, it is by alcohol oxidase promotor pAOX1 and pAOX2 from methanol yeast, Protosol synthase promoter pDAS, P40 gene promoter pP40, the catalase promotor, formate dehydrogenase promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter pGAP and formaldehyde dehydrogenase promotor pFLD, and from yeast Candida, the promotor of two or more that the activation expression of selecting in the eukaryotic promoter of Hansenula and Torulopsis does not conflict, for example two kinds of promotors of pAOX1 and pGAP are built into the exogenous gene expression box respectively.Then be integrated in the same methanol yeast host cell, the synergistic effect by multiple promoter efficiently expresses foreign protein, shortens fermentation time simultaneously, and makes expression condition oversimplify.

Foreign gene comprises human serum albumin (HSA), horseradish peroxidase, p53 albumen, thyrocalcitonin or the like, is research object with human serum albumin (HSA) below, and the example that contains two kinds of many expression cassettes of promotor is provided.The gene of human serum albumin (HSA) can be by the method for reverse transcription PCR, and reverse transcription obtains from people's embryo liver cell.Also can adopt total length synthetic method, use the synthetic complete HSA gene order of dna synthesizer.The present invention adopts the method for reverse transcription PCR to obtain the HSA full-length cDNA, its dna sequence dna front end contains natural HSA secreting signal peptide and job sequence (Fig. 1, wherein 54～125bp is coding 24 amino acid whose human serum albumin secretion job sequence, i.e. prepro sequences; 126～1883bp is for becoming acquaintance HSA encoding sequence).According to the operation of conventional molecular biology, the HSA gene that obtains is packed among methanol yeast expression plasmid pPIC3.5K and the pGAPZ α A, obtain HSA yeast recombinant expression plasmid pPIC3.5K-HSA (Fig. 2) and pGAP-HSA (Fig. 4) respectively.Among Fig. 2, the HSA gene is positioned under the pAOX1 promotor, and the site of packing into is BamH I and EcoR I, contains kanamycin multiple copied resistance screening mark in the plasmid.And among Fig. 4, the HSA gene is positioned under the pGAP promotor, and the site of packing into is NspV and the EcoRI site among the pGAPZ α A, contains Zeocin multiple copied resistance screening mark in the plasmid, but this recombinant expression plasmid constitutive expression HSA.Above-mentioned two kinds of HSA yeast recombinant expression plasmids are integrated into same methanol yeast host cell, obtain two kinds of many expression cassettes of the promotor engineering strains that contain of HSA.Wherein methanol yeast Pichia pastoris host cell can be GS115, KM71, SMD1168, SMD1168H etc., and the method for integrating the methanol yeast cell can be ball plastid method and electroporation conversion method etc.

Contain different resistance screening mark (Kanamycin and Zeocin) based on the yeast recombinant expression plasmid pPIC3.5K-HSA that makes up with pGAP-HSA, therefore making up when this contains two kinds of many expression cassettes of promotor engineering strains to adopt two kinds of recombinant expression plasmids to transform simultaneously, two kinds of resistance while method for screening are carried out, also can earlier a kind of recombinant expression plasmid be integrated in the yeast host cell, by corresponding resistance screening positive colony, then the positive colony that obtains with screening again is a host cell, import another kind of recombinant expression plasmid, go out to be integrated with many expression cassettes engineering strain of two kinds of recombinant expression plasmids by the resistance screening of recombinant expression plasmid correspondence therewith.The present invention adopts a kind of method in back to carry out, and the yeast host cell that uses is GS115.What the result was constructed contains two kinds of many expression cassettes of promotor HSA engineering strains without methanol induction the time, can be by containing a plurality of expression cassette constitutive expression HSA of pGAP promotor.And a plurality of expression cassettes that contain pGAP and two kinds of promotors of pAOX1 behind methanol induction can act on simultaneously, and its expression amount is apparently higher than the engineering strain that only contains single promoter expression cassettes.

In the structure example that more than provides, the constructed HSA engineering strain that contains pGAP and two kinds of many expression cassettes of promotor of pAOX1, its HSA expression amount obviously improves.If the promotor in the many expression cassettes of multiple promotor includes pAOX1, also can be at the characteristic of pAOX1 promotor, by following improvement, when improving expression amount to be implemented in, the fermentation character of optimization expression bacterial strain.

Exist the expression foreign protein to carry out stage by stage at the engineering strain that has the pAOX1 promotor to make up, shortcomings such as the cycle is long, fermentation condition is wayward can be improved the characteristic that this type of includes many expression cassettes of multiple promoter engineering strain of pAOX1 by the method for mutagenesis.Wherein mutagenesis can be adopted the method for uv irradiating, also can be undertaken by using mutagenic compound.Mutagenic compound commonly used can be: EMS, 2-aminopurine, azanol, N-methyl-N '-nitro-N-nitrosoguanidine, DES and bifurcation pyridine etc.Foreign gene still is example with the human serum albumin.At first make up HSA yeast recombinant expression plasmid pPIC3-HSA (Fig. 3), be integrated among the yeast host cell GS115, filter out the HSA cloning by expression, then adopt the EMS mutagenic compound, screening obtains HSA engineering strain (Fig. 5 that a methanol induction is not suppressed by glucose, show that wherein non-mutagenic strain do not express HSA in containing dextrose culture-medium, and mutagenic strain can be expressed).Then with this mutagenic obtained HSA expression strain as host cell, be integrated into HSA composing type recombinant expression plasmid pGAP-HSA (Fig. 4), through expression screening, the result obtains the good engineering strain Pichia pastoris SIB 121 of a HSA expression characterization (be deposited in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms center on July 15th, 1999, deposit number is CGMCC NO.0407).This engineering strain is because the methanol induction characteristic of pAOX1 no longer is subjected to the inhibition of carbon sources such as glucose, therefore can methanol induction and the cell growth carry out simultaneously, promptly no longer need to ferment stage by stage, reached the purpose that shortens fermentation time.Simultaneously, because the methanol induction characteristic of pAOX1 no longer is subjected to the inhibition of carbon sources such as glucose and glycerine, so does not exist the control process of complexity such as glycerine vegetative period-glycerine phase of limiting the quantity of-methanol induction phase yet in the large scale fermentation, simplified the control of fermentation condition.And by making up the many expression cassettes of multiple promotor, its expression amount is more apparently higher than the constructing system (Fig. 6, Fig. 7) of single promotor.

By above example as can be seen, by methanol yeast Pichia pastoris host cell GS115 is carried out mutagenesis with the EMS mutagenic compound, the host cell that the methanol induction that obtains a pAOX1 is not suppressed by carbon source such as glucose, then, contain the many expression cassettes of multiple promotor with this host cell in conjunction with what the present invention set up, can in methanol yeast, express foreign protein more easily.Thereby increase work efficiency.

Advantage of the present invention and effect:

The many expression cassettes of multiple promotor that contain in the methanol yeast Pichia pastoris expression system that the present invention sets up, good characteristic that can the multiple source of polymerization eukaryotic promoter, in constructed gene engineering expression bacterial strain, improve its characteristic jointly and improve the expression amount of recombinant exogenous protein, avoided the good but not high shortcoming of expression amount of some single promoter expression cassettes bacterial strain expression characterization.

When including the pAOX1 promotor in the many expression cassettes of multiple promotor that make up, can improve the characteristic that this type of includes many expression cassettes of multiple promoter engineering strain of pAOX1 by the method for mutagenesis, the engineering strain that make to make up methanol induction and cell growth during the fermentation carries out simultaneously, shortened fermentation time, simplify fermentation condition, improved the expression amount of foreign protein simultaneously by multiple many expression cassettes of promotor constructing system.Reduced the cost of industrial generation on the whole.

Description of drawings

Fig. 1, people's embryo liver preproHSAcDNA sequence.

The design of graphics of Fig. 2, HSA yeast recombinant expression plasmid pPIC3.5K-HSA.

The design of graphics of Fig. 3, HSA yeast recombinant expression plasmid pPIC3-HSA.

Fig. 4, HSA yeast recombinant expression plasmid pGAP-HSA design of graphics.

The SDS-PAGE electrophorogram of Fig. 5, the non-inhibition of glucose HSA engineering bacterium expression HSA.(1. lower molecular weight standard protein; 2. mutagenesis starting strain, in contrast; Produce the HSA engineering strain 3-7. be respectively the non-inhibition type of the glucose of screening).

Fig. 6, multiple many expression cassettes of promotor bacterial strain SIB 121 and single promotor bacterial strain GS115/pPIC3-HSA ⁺The SDS-PAGE electrophoresis of expressing HSA is (1. lower molecular weight standard protein relatively; 2.SIB121 in BMMGY, induce two days supernatant liquors, last sample 10 μ l; 3.GS115/pPIC3-HSA ⁺In BMMGY, induce two days supernatant liquors, last sample 10 μ l; 4.SIB 121 induce two days supernatant liquors in BMGY, last sample 10 μ l).

Fig. 7, multiple many expression cassettes of promotor bacterial strain SIB 121 and single promotor bacterial strain GS115/pPIC3-HSA ⁺(1.SIB 121 induces two days supernatant liquors to the SDS-PAGE scanning analysis of expression HSA in BMMGY, last sample 10 μ l; 2.GS115/pPIC3-HSA ⁺In BMMGY, induce two days supernatant liquors, last sample 10 μ l; 3.SIB 121 induce two days supernatant liquors in BMGY, last sample 10 μ l).

The present invention can further set forth by following embodiment, but does not limit the scope of the invention.Experiment material explanation: (1) reagent

The enzyme of DNA cloning test kit, klenow fragment polymerase and all uses is GIBCO BRL company product.Determined dna sequence test kit, Trizol RNA extraction agent box are available from Promega company.YNB (w/o amino acid) is available from DIFCO company, and G418 and Zeocin are available from GIBCO BRL company.It is Promega company product that Wizard plus minipreps DNA plasmid extraction test kit, Wizard PCR preps DNA reclaim test kit.This laboratory of HSA antiserum(antisera) provides.Electricity conversion instrument CELL-PORATOR is a GIBCOBRL company product.(2) plasmid and bacterial classification

Plasmid pPIC3, pPIC3.5K and pGAPZ α A, methanol yeast bacterial strain Pichia pastorisGS115 (his4Mut ⁺) being Invitrogen company product, plasmid PUC19 and E.Coli DH5 α F ' bacterial strain are the preservation of inventor laboratory.

The clone of embodiment 1 preproHSA cDNA

Get 2 gram people embryo liver cells, according to Trizol RNA extraction agent box recommend method, extracted total RNA from people's embryo liver cell.According to known natural HSA gene 5 ' and 3 ' terminal sequence, the design primer is as follows:

Primer 1:

Primer 2:

Total RNA is a template with extractive people's embryo liver cell, and human serum albumin preproHSA cDNA is synthesized in the PCR reverse transcription.Reaction conditions is: 94 ℃ of sex change 45 seconds, and 55 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute and 30 seconds, totally 40 circulations, last 72 ℃ of insulations 10 minutes.The PCR product is after the preliminary conclusive evidence of 1% agarose electrophoresis, and is according to the recommendation using method of the GIBCO BRL Klenow of company polymerase that the PCR product is flat with Klenow fragment polysaccharase benefit, and adopts Promega DNA to reclaim test kit and reclaim the flat pcr amplified fragment of benefit.

Be connected with the PUC19 carrier flush end that the SmaI enzyme is cut mending flat PCR product, the ligation system is: PUC19 (SmaI cuts) 1 μ l, 5 * connection damping fluid, 3 μ l, T4 dna ligase (1U/ μ l) 1.5 μ l, SmaI (1U/ μ l) 0.5 μ l, PCR mends the thing 5 μ l that show no increases in output, sterilized water 4 μ l.Above mixture connects 5 hours for 21 ℃.Gained connects product Transformed E .coli DH5 α F ' competent cell, coating ampicillin plate screening positive clone.Adopt Promega plasmid extraction test kit to prepare plasmid DNA.The gained plasmid DNA goes out recombinant plasmid PUC19-HSA with BamHI and EcoRI double digestion preliminary evaluation.Dna sequence analysis shows (Fig. 1): the Chinese HSA gene of pcr amplification gained and the basically identical of bibliographical information, only 122 Arg (AGA) at the preproHSA peptide sequence become Ser (AGT), 466 Glu (GAA) become Gly (GGA), and 162 changes (TTA becomes CTA) do not cause amino acid whose variation.

Embodiment 2 HSA yeast recombinant expression plasmid pPIC3.5K-HSA and pPIC3-HSA make up

PreproHSA gene fragment in the PUC19-HSA cloning vector is used under BamH I and the EcoR I double digestion, 1% agarose electrophoresis, Promega PCR Prep Kit reclaims the preproHSA gene fragment of about 1.8Kb.Reclaim fragment and be connected with the pPIC3.5K expression plasmid of cutting with same enzyme, the ligation system is: pPIC3.5K (BamHI, EcoRI is two to be cut) 1 μ l, 5 * connection damping fluid, 3 μ l, T4 dna ligase (1U/ μ l) 1.5 μ l, preproHSA 3 μ l, sterilized water 6.5 μ l.Above mixture connects 5 hours for 21 ℃.Gained connects product Transformed E .coli DH5 α F ' competent cell, coating ampicillin plate screening positive clone.Promega plasmid DNA extraction agent box prepares plasmid DNA.The gained plasmid DNA with BamHI and EcoRI double digestion identify recombinant expression plasmid pPIC3.5K-HSA (Fig. 2).

Same quadrat method, the recombinant expression plasmid pPIC3.5K-HSA of above-mentioned structure is cut and reclaims the HSA gene fragment through SacI and SalI enzyme, be connected with the expression plasmid of yeast pPIC3 that cuts with same enzyme, enzyme is cut checking can screen acquisition yeast HSA recombinant expression plasmid pPIC3-HSA (Fig. 3).

The structure of embodiment 3 HSA yeast composing type recombinant expression plasmid pGAP-HSA

The pPIC3.5K-HSA recombinant expression plasmid DNA that makes up is cut with the BamHI enzyme, then handle 15 minutes removal BamHI enzymes with s1 nuclease in 23 ℃ and cut the sticky end of generation, PromegaDNA reclaims test kit and reclaims linear DNA.Gained blunt end linear DNA again with the EcoRI enzyme cut, 1.5% low melting-point agarose electrophoresis, glue reclaims the preproHSA gene fragment of about 1.8Kb.PGAPZ α A expression plasmid after the NspV enzyme is cut, by above same procedure with s1 nuclease remove that sticky end, EcoRI enzyme are cut, 1% low melting-point agarose electrophoresis and reclaim the 2.9Kb fragment.The preproHSA gene fragment in gained linearizing dna fragmentation and aforementioned pPIC3.5-HSA source is carried out ligation, its system is: pGAPZ α A (NspV+S1+EcoRI) 2 μ l, preproHSA (BamH I+S1+EcoR I) 8 μ l, 5 * connection damping fluid, 3 μ l, T4 dna ligase (1U/ μ l) 2 μ l.16 ℃ of connections of above mixture are spent the night.Gained connects product Transformed E .coli DH5 α F ' competent cell, coating SLB/Zeocin flat board (0.5% yeast extract paste, 1% peptone, 0.5%NaCl, 25mg/l Zeocin) screening positive clone.Promega plasmid DNA extraction agent box prepares plasmid DNA.The gained plasmid DNA with Bgl II and EcoR I double digestion identify composing type recombinant expression plasmid pGAP-HSA (Fig. 4).

Embodiment 4 pPIC3.5k-HSA electricity transformed yeast cell and expression thereof

With the about 10 μ g of the recombinant expression plasmid pPIC3.5K-HSA Sal I linearization for enzyme restriction that makes up, ethanol sedimentation reclaims linear DNA and also is dissolved in the 10 μ l sterilized waters, simultaneously the identical linearization for enzyme restriction of unloaded pPIC3.5K plasmid is also reclaimed in contrast.GS115 is in 500ml YPD substratum (1% yeast extract paste, 2% peptone, 2% glucose) in inoculation, and 28-30 ℃ is cultured to OD ₆₀₀Be 1.3-1.5, the centrifugal 5min of 5000r/min, bacterial sediment is respectively washed once with the ice-cold sterilized water of 100ml, the ice-cold sterilized water of 20ml and the ice-cold sorbyl alcohol of 20ml1mol/l respectively, wash the equal centrifugal 5min of 5000r/min in back at every turn and collect thalline, at last the centrifugal somatic cells is suspended with the ice-cold sorbyl alcohol of 200 μ l 1M, promptly obtain the electric shock competent cell.Above-mentioned linearizing DNA is mixed with 80 μ l GS115 electricity transformant respectively, adopt GIBCOBRL electricity conversion instrument CELL-PORATOR electric shock to transform, electric conversion condition is: voltage 1500V, electric capacity 50 μ F, resistance 4K Ω.The electricity converted product is transferred to respectively in the aseptic Eppendorf tube, the sorbyl alcohol that adds the precooling of 0.50mL1mol/l ice bath immediately, 30 left standstill one hour, 30 ℃ of 200rpm cultivated 1-1.5 hour after adding 0.5ml YPD, quick centrifugal removal supernatant adds 300 μ l sterilized water suspension thalline, respectively gets the YPD flat board (containing each 0.5mg/ml of G418,1.0mg/ml and 1.5mg/ml) that 100 μ l coating contains different concns G418,30 ℃ connect MD flat board (13.4g/l YNB, 4 * 10 with the positive colony point after 2～4 days ^-4The g/l vitamin H, 20g/l glucose, 20g/l agar) checking his+ phenotype, 30 ℃ of positive colonies after 2 days are Pichia pastoris GS115/HSA (his+Mut+) recombinant clone, and the positive colony that wherein contains on the high YPD flat board of G418 concentration is the multiple copied recombinant clone.

HSA engineering strain (GS115/pPIC3.5k-HSA) inoculation 3ml YPD test tube is produced in the strain that above-mentioned structure screening obtains, overnight incubation in 30 ℃ of 300r/min shaking tables, contain 8mlBMGY (10g/l yeast extract paste with the 0.1-0.2% inoculation, the 20g/l peptone, 0.1mol/l potassium phosphate buffer pH6.0,13.4g/lYNB, 4 * 10 ^-4G/l vitamin H, 10g/l glycerine) in the 50ml culture tube.30 ℃ of 300r/min are cultured to OD ₆₀₀Be 2～10.The centrifugal 5-10min of normal temperature 5000r/min.Collected thalline is transferred to the 250ml triangular flask after suspending with 40mlBMMY (10g/l glycerine among the BMGY is changed into 5ml/l methyl alcohol), and 28 ℃ of 300r/min begin to induce.Added methyl alcohol to 5ml/l in per 24 hours.Induce after 48 hours and detect fermented liquid supernatant with self-control HSA antiserum(antisera) ELISA, the HSA expression amount is about 60-80mg/l.

The non-inhibition type of embodiment 5 glucose HSA engineering bacteria GS115/pPIC3-HSA ⁺Acquisition

The HSA recombinant expression plasmid pPIC3-HSA that embodiment 2 is obtained presses embodiment 4 described step electricity transformed yeast cell GS115, then is coated with the MD flat board, cultivates after 2-4 days for 30 ℃, and positive colony is through MM flat board (13.4g/l YNB, 4 * 10 ^-4G/l vitamin H, 5ml/l methyl alcohol, 20g/l agar) checking, select to such an extent that methyl alcohol utilizes the HSA cloning by expression (GS115/pPIC3-HSA) of normal type.

The HSA cloning by expression GS115/pPIC3-HAS that obtains is seeded to 3ml YPD test tube, 28 ℃, 300r/min overnight incubation.Get the centrifugal 1min of 0.6ml bacterium liquid normal temperature 15000r/min.Collect thalline, behind the sterilized water repetitive scrubbing, be resuspended in 2ml sodium phosphate buffer (pH7.0).Add 60 μ lEMS stostes, after mixing, incubation 1hr on 28 ℃ of rotary shakers.With 400 times of aseptic 5%Na of volume ₂S ₂O ₃Handle deactivation EMS.The centrifugal 1min of normal temperature 15000r/min collects thalline, is suspended in the 2ml sterilized water.Be coated with the bm-YNB flat board (0.1mol/l phosphoric acid buffer pH6.0,5ml/l methyl alcohol, 13.4g/lYNB, 15g/l agar) that contains the 0.02%2-deoxyglucose with different concns, put 30 ℃ and cultivated 2-5 days.The gained positive colony is seeded to 3ml YPD test tube, 28-30 ℃, 300r/min overnight incubation.1% is forwarded to 10ml YPDM (10g/l yeast extract paste, 20g/l peptone, 20g/l glucose, 5ml/l methyl alcohol), and 28-30 ℃, 300r/min were induced 2-3 days, and adding methyl alcohol to final concentration every day is 5ml/l.Fermented liquid supernatant shows through the SDS-PAGE electrophoresis, EMS mutagenic strain (GS115/pPIC3-HSA ⁺) than mutagenic strain (GS115/pPIC3-HSA) not tangible 67kD protein band (Fig. 5) is arranged.

Embodiment 6 contains two kinds of many expression cassettes of promotor HSA engineering bacteria GS115/pPIC3.5k+p GAP

With the about 10 μ g of the recombinant expression plasmid pGAP-HSA PshA I linearization for enzyme restriction that embodiment 3 makes up, ethanol sedimentation reclaims linear DNA and also is dissolved in the 10 μ l sterilized waters.Experimental procedure with embodiment 4 prepares GS115/pPIC3.5k-HSA electric shock competent cell.Above-mentioned linearizing DNA is mixed with 80 μ l electricity transformant respectively, adopt GIBCO BRL electricity conversion instrument CELL-PORATOR, transform with embodiment 4 electric shock step of converting electric shocks.Get YPDS flat board (10g/l yeast extract paste, 20g/l peptone, 20g/l glucose, 1mol/l sorbyl alcohol, 20g/l agar that the coating of 100 μ l transformants contains different concns Zeocin at last, Zeocin concentration is 100mg/l or 500mg/l), cultivated 2～4 days for 30 ℃, the gained positive colony is the engineering bacteria that contains pAOX1 and two kinds of promoter expression HSA of pGAP.The positive colony that wherein contains on the high YPDS flat board of Zeocin concentration is a pGAP-HSA multiple copied recombinant clone.Obtain a strain by the last screening of above step and contain two kinds of many expression cassettes of promotor HSA engineering bacteria GS115/pPIC3.5k+p GAP, this bacterial strain can be expressed HSA in substratum YPD that does not contain methyl alcohol or BMGY, show that composing type recombinant expression plasmid pGAP-HSA can express HSA in yeast.Through the ELBA assay determination, this bacterial strain methanol induction after 2 days the HSA expression amount can reach about 120mg/l.

Embodiment 7 contains the structure of two kinds of many expression cassettes of promotor HSA engineering strain Pichia pastoris SIB 121

With embodiment 6 steps, with the HSA bacterial strain GS115/pPIC3-HSA that obtains among the embodiment 5 ⁺Be host cell, electricity is transformed into composing type recombinant expression plasmid pGAP-HSA, through inducing screening, obtains an engineering bacteria GS115/pAOX1+pGAP (Pichia pastoris SIB 121).This engineering bacteria can be in YPDM or BMMGY (10g/l yeast extract paste, 20g/L peptone, 0.1mol/l potassium phosphate buffer pH6.0,13.4g/lYNB, 4 * 10 ^-4G/l vitamin H, 10g/l glycerine, 5ml/l methyl alcohol) start two promoter expression HSA simultaneously in the substratum, shake flask fermentation liquid is through the SDS-PAGE analysis revealed, and its expression amount is apparently higher than the engineering bacteria (Fig. 6, Fig. 7) of single promotor.Carry out elisa assay with self-control HSA antiserum(antisera), show that SIB 121 just can reach nearly 120mg/l (shaking bottle) through two days its HSA expression amounts of BMMGY fermentation.

Claims (4)

1. many expression cassettes of multiple promotor that contain in the methanol yeast expression system, it is characterized in that described multiple promotor is by alcohol oxidase promotor pAOX1 and pAOX2 from methanol yeast, Protosol synthase promoter pDAS, P40 gene promoter pP40, the catalase promotor, formate dehydrogenase promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter pGAP and formaldehyde dehydrogenase promotor pFLD, and from yeast Candida, the promotor of two or more that the activation expression of selecting in the eukaryotic promoter of Hansenula and Torulopsis does not conflict, be built into the exogenous gene expression box respectively, then be integrated in the same host cell.

2. the many expression cassettes of multiple promotor that contain according to claim 1 is characterized in that the described many expression cassettes that contain multiple promotor are the many expression cassettes that contain pAOX1 and two kinds of promotors of pGAP.

3. a class contains the engineering strain of the many expression cassettes of the described multiple promotor of claim 1, it is characterized in that to be integrated into by the exogenous gene expression box that multiple promotor is built into respectively in the same methanol yeast host cell foreign gene engineering strain that contains the many expression cassettes of multiple promotor that obtains through expression screening.

4. engineering strain according to claim 3, it is characterized in that pAOX1 and two kinds of promotors of pGAP are made up the human serum albumin gene expression cassette respectively, be integrated in the same yeast host cell and obtain deposit number contains the many expression cassettes of multiple promotor for CGMCC NO.0407 human serum albumin gene engineering strain Pichia pastoris SIB 121 through expression screening.

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