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

Abstract

The present invention improves the existing methanol yeast expression system and provides multiple promoter multi-expression cassettes contained in the methanol yeast expression system. By looking for promoters that do not conflict with each other in terms of activation and expression, the expression cassettes of foreign genes are constructed separately, and after being integrated into the same yeast cell, a genetically engineered strain integrated with multiple expression cassettes containing multiple promoters is obtained, and the expression of foreign proteins is obvious. Higher than single promoter expression cassette engineering strains. Through mutagenesis or selection of a suitable promoter such as constitutive type, growth and expression can be achieved simultaneously, the fermentation time can be shortened, and the fermentation conditions can be simplified.

Description

Multiple promoter multiple expression cassettes contained in the methanolic yeast expression system

The invention relates to a methanol yeast eukaryotic expression system, in particular to the high-efficiency expression of foreign proteins in the methanol yeast eukaryotic expression system by using a multi-expression cassette containing multiple promoters.

The methanolic yeast Pichia pastoris expression system is a eukaryotic expression system successfully developed by Invitrogen. Due to its high expression level, secreted expression, and less foreign proteins, a large number of foreign proteins have been successfully expressed in the methanol yeast system in recent years (Ohtani W, Ohda T, Sumi A et al. Anal Chem 70: 425～ 429, 1998). The methanolic yeast expression plasmid generally contains: foreign protein expression promoter, multiple cloning site, 3' terminal transcription termination sequence (AOX1 3' terminal sequence), and the promoter and replicator required for the expression of resistance genes. For secretory plasmids, there is also a secretory signal peptide sequence between the promoter and the multiple cloning site. The foreign gene is inserted into the multiple cloning site behind the promoter or signal peptide. The promoter sequence or 3' end sequence in the expression plasmid integrates the exogenous gene expression cassette into the yeast chromosome through homologous recombination with the Pichia pastoris chromosomal DNA, and induces the expression of the exogenous gene by corresponding to the promoter, so The choice of promoter has a very important impact on the expression of foreign proteins.

Most of the promoters that can be used for the expression of methanolic yeast are derived from methanolic yeast itself, such as pAOX (pAOX1 and pAOX2), pDAS, pP40, pGAP and pFLD, etc. Different promoters have different activating transcription characteristics according to the role of their naturally transcribed proteins in yeast, and their strengths are also different. According to the existing reports on the expression system of Pichia pastoris, the alcohol oxidase promoter pAOX1 is the most used one with high expression efficiency. pAOX1 is a strong promoter, and the foreign protein expression engineering bacteria constructed by using it can be induced by methanol, and a high expression level can be obtained in shake flasks. The pGAP and pFLD promoters were recently reported (Shen-S, et al. Gene216 (1): 93-102, 1998; Doring-F, et al. Biochem Biophys Res Commun, 250 (2): 531 -535, 1998), according to Doring et al. reported that in the Pichia pastoris expression system, the pGAP promoter is more suitable for expressing functional mammalian transporters than pAOX1. In addition, eukaryotic promoters derived from Candida, Hansenula and Torulopsis can also be used for exogenous gene expression in methanolic yeast.

However, the existing patents and related documents show (US 5643792; H.R.Waterham, et alGene 186(1):37-44, 1997), the pAOX1 expression system that is now widely used is due to the toxicity of methanol to cells and the toxicity of glycerol to methanol Induced weak inhibition and other reasons, in the fermenter must first go through a 1-2 day glycerol growth period, then add glycerol in a limited amount to make the cells grow to a suitable density, and finally add methanol gradually to induce expression, the whole cycle is as long as 7-12 days, and this method of inducing expression is difficult to control, so the high efficiency of the pAOX1 promoter is often not fully displayed. Although promoters such as pGAP can constitutively express foreign proteins, for most foreign proteins, the expression level after using the pGAP promoter is not satisfactory, which makes its application far less extensive than pAOX1.

The most successful example of the methanolic yeast expression system is the high-efficiency expression of human serum albumin (Human Serum Albumin, HSA) in the methanolic yeast Pichia pastoris. HSA is an important clinical drug with great demand. Due to the spread of diseases such as AIDS and hepatitis, blood-derived HSA drugs have the threat of carrying viruses, and related accidents have been reported from time to time. Therefore, for 15 years, the international community has been exploring the research and development of genetically engineered HSA drugs, among which the expression system of methanolic yeast Pichia pastoris is the most successful one. The expression of HSA in methanolic yeast is an exocrine type, the promoter used is pAOX1, and the expression level of the shake flask is about 50mg/l. Related patents include US5707828, EP 0570916A2, EP 0510693A2, etc. Therefore, it is of great application value to establish a new multi-expression cassette construction system containing multiple promoters in methanolic yeast, so that the high-efficiency expression of foreign proteins can be obtained by adopting simpler and more convenient fermentation conditions.

The purpose of the present invention is to improve the existing expression system of methanolic yeast Pichia pastoris, and provide multiple promoter multi-expression cassettes contained in the methanolic yeast expression system, so that the expression of exogenous protein can be obtained by adopting simpler and more convenient fermentation conditions Efficient expression reduces the cost of industrial production as a whole.

The present invention provides a multi-promoter multi-expression cassette contained in a methanolic yeast expression system, which consists of alcohol oxidase promoters pAOX1 and pAOX2, dihydroxyacetone synthase promoter pDAS, and P40 gene promoter pP40 from methanolic yeast , catalase promoter, formate dehydrogenase promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter pGAP and formaldehyde dehydrogenase promoter pFLD, and eukaryotic promoters from yeast Candida, Hansenula and Torulopsis Two or more kinds of promoters selected from among those whose activation and expression do not conflict, such as pAOX1 and pGAP promoters, are respectively constructed into exogenous gene expression cassettes. Then it is integrated into the same methanolic yeast host cell, and the superimposed effect of multiple promoters enables the high-efficiency expression of the foreign protein, shortens the fermentation time, and simplifies the expression conditions.

Exogenous genes include human serum albumin (HSA), horseradish peroxidase, p53 protein, calcitonin, etc. The following takes human serum albumin (HSA) as the research object, and provides multiple expression cassettes containing two promoters instance of . The gene of human serum albumin (HSA) can be obtained by reverse transcription from human embryonic liver cells by reverse transcription PCR. The method of full-length synthesis can also be adopted, and a complete HSA gene sequence can be synthesized using a DNA synthesizer. The present invention adopts the method of reverse transcription PCR to obtain the full-length cDNA of HSA, and the front end of the DNA sequence contains a natural HSA secretion signal peptide and a processing sequence (Fig. That is the prepro sequence; 126~1883bp is the mature human HSA coding sequence). According to conventional molecular biology operations, the obtained HSA gene was loaded into methanolic yeast expression plasmids pPIC3.5K and pGAPZαA to obtain HSA yeast recombinant expression plasmids pPIC3.5K-HSA (Fig. 2) and pGAP-HSA (Fig. 4), respectively. In Figure 2, the HSA gene is located under the pAOX1 promoter, the loading sites are BamHI and EcoRI, and the plasmid contains a multi-copy kanamycin resistance selection marker. In Figure 4, the HSA gene is located under the pGAP promoter, the loading site is the NspV and EcoRI sites in pGAPZαA, and the plasmid contains Zeocin multi-copy resistance selection marker, and this recombinant expression plasmid can express HSA constitutively. The above two HSA yeast recombinant expression plasmids are integrated into the same methanol yeast host cell to obtain a genetically engineered strain of HSA containing two kinds of promoters with multiple expression cassettes. Among them, the host cells of methanolic yeast Pichia pastoris can be GS115, KM71, SMD1168, SMD1168H, etc., and the method of integrating methanolic yeast cells can be spheroplast method and electroporation transformation method.

Based on the construction of yeast recombinant expression plasmids pPIC3.5K-HSA and pGAP-HSA contain different resistance selection markers (Kanamycin and Zeocin), so two kinds of recombinant expression plasmids can be used to construct this multi-expression cassette engineering strain containing two kinds of promoters Simultaneous transformation and simultaneous screening of two resistances can also be carried out by first integrating a recombinant expression plasmid into yeast host cells, screening positive clones through corresponding resistance, and then using the positive clones obtained by screening as host cells , introduce another recombinant expression plasmid, and select the multi-expression cassette engineering strain integrated with the two recombinant expression plasmids through the resistance corresponding to the recombinant expression plasmid. The present invention adopts the latter method, and the yeast host cell used is GS115. Results The constructed HSA genetically engineered strain containing two kinds of promoter multiple expression cassettes could constitutively express HSA through multiple expression cassettes containing pGAP promoter without induction by methanol. However, multiple expression cassettes containing two promoters of pGAP and pAOX1 can act simultaneously after being induced by methanol, and its expression level is significantly higher than that of the genetically engineered strain containing only a single promoter expression cassette.

In the construction examples provided above, the constructed HSA engineering strains containing pGAP and pAOX1 promoter multiple expression cassettes have significantly increased expression of HSA. If the promoters in the multi-promoter multi-expression cassette contain pAOX1, the following improvements can be made according to the characteristics of the pAOX1 promoter, so as to increase the expression amount and optimize the fermentation characteristics of the expression strain.

In view of the shortcomings of the genetically engineered strains constructed with the pAOX1 promoter, the expression of foreign proteins must be carried out in stages, the cycle is long, and the fermentation conditions are not easy to control. Mutagenesis can be used to improve this type of multi-promoter multi-expression cassette containing pAOX1 Characteristics of genetically engineered strains. The mutagenesis can be carried out by means of ultraviolet irradiation or by using a mutagen. Commonly used mutagens can be: EMS, 2-aminopurine, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, DES and acridine, etc. The exogenous gene still takes human serum albumin as an example. First construct the HSA yeast recombinant expression plasmid pPIC3-HSA (Figure 3), integrate it into the yeast host cell GS115, screen out the HSA expression clone, and then use the EMS mutagen to screen to obtain a methanol-induced HSA engineering strain that is not inhibited by glucose ( Figure 5, which shows that the non-mutated strains do not express HSA in glucose-containing medium, while the mutagenized strains can express it). Then the HSA expression strain obtained by this mutagenesis was used as a host cell, and integrated into the HSA constitutive recombinant expression plasmid pGAP-HSA (Figure 4). After expression screening, a genetically engineered strain Pichia pastoris SIB 121 with excellent HSA expression characteristics was obtained ( On July 15, 1999, it was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee in Beijing, and the preservation number is CGMCC NO.0407). Since the methanol-inducible properties of pAOX1 are no longer inhibited by carbon sources such as glucose, the engineered strain can be induced by methanol and cell growth at the same time, that is, it is no longer necessary to carry out staged fermentation, and the purpose of shortening the fermentation time is achieved. At the same time, because the methanol-inducible properties of pAOX1 are no longer inhibited by carbon sources such as glucose and glycerol, there is no complex control process such as glycerol growth period-glycerol limit period-methanol induction period in large-scale fermentation, which simplifies the adjustment of fermentation conditions. control. However, by constructing a multi-expression cassette with various promoters, its expression level is significantly higher than that of a single-promoter construction system (Fig. 6, Fig. 7).

As can be seen from the above examples, by mutagenizing the methanolic yeast Pichia pastoris host cell GS115 with an EMS mutagen, a host cell in which the methanol induction of pAOX1 is not inhibited by carbon sources such as glucose is obtained, and then, the host cell is used to bind this The multi-expression box containing multiple promoters established by the invention can more easily express foreign proteins in methanolic yeast. Thereby improving work efficiency.

Advantages and effects of the present invention:

The various promoter multi-expression cassettes contained in the methanolic yeast Pichia pastoris expression system established by the present invention can aggregate the excellent characteristics of eukaryotic promoters from various sources, and jointly improve their characteristics and enhance the recombination in the constructed genetic engineering expression strains. The expression level of the foreign protein avoids the disadvantage of some single-promoter expression cassette strains with good expression characteristics but low expression level.

When the multi-promoter multi-expression cassette of the construction contains the pAOX1 promoter, the method of mutagenesis can be used to improve the characteristics of this type of multi-promoter multi-expression cassette engineering strain comprising pAOX1, so that the engineered strain of construction can be used in During the fermentation process, methanol induction and cell growth are carried out simultaneously, which shortens the fermentation time and simplifies the fermentation conditions. At the same time, the expression of foreign proteins is increased through the construction of multiple promoters and multiple expression cassettes. The cost of industrial production is reduced as a whole.

Description of drawings

Figure 1. Human embryonic liver preproHSAcDNA sequence.

Fig. 2. The construction diagram of the HSA yeast recombinant expression plasmid pPIC3.5K-HSA.

Fig. 3. The construction diagram of the HSA yeast recombinant expression plasmid pPIC3-HSA.

Fig. 4. The construction diagram of the HSA yeast recombinant expression plasmid pGAP-HSA.

Fig. 5. SDS-PAGE electrophoresis of glucose non-inhibited HSA engineered bacteria expressing HSA. (1. Low-molecular-weight standard protein; 2. The starting strain for mutagenesis, as a control; 3-7. Respectively, screened non-inhibitory glucose-producing HSA engineering strains).

Figure 6. Comparison of SDS-PAGE electrophoresis between multiple promoter multi-expression cassette strain SIB 121 and single promoter strain GS115/pPIC3-HSA ⁺ expressing HSA (1. Low molecular weight standard protein; 2. Supernatant of SIB121 induced in BMMGY for two days 3. The supernatant of GS115/pPIC3-HSA ⁺ induced in BMMGY for two days, loaded in 10 μl; 4. The supernatant of SIB 121 induced in BMGY for two days, loaded in 10 μl).

Figure 7. SDS-PAGE scanning analysis of multiple promoter multi-expression cassette strain SIB 121 and single promoter strain GS115/pPIC3-HSA ⁺ expressing HSA (1. SIB 121 was induced in BMMGY for two days supernatant, loading 10 μl; 2. The supernatant of GS115/pPIC3-HSA ⁺ induced in BMMGY for two days, loaded 10 μl; 3. The supernatant of SIB 121 induced in BMGY for two days, loaded with 10 μl).

The present invention can be further illustrated by the following examples, which do not limit the scope of the present invention. Description of experimental materials: (1) Reagents

DNA amplification kit, klenow fragment polymerase and all enzymes used are products of GIBCO BRL company. DNA sequence determination kit and Trizol RNA extraction kit were purchased from Promega. YNB (w/o amino acid) was purchased from DIFCO, G418 and Zeocin were purchased from GIBCO BRL. Wizard plus minipreps DNA plasmid extraction kit and Wizard PCR preps DNA recovery kit are products of Promega. HSA antiserum was provided by this laboratory. The electric conversion instrument CELL-PORATOR is a product of GIBCOBRL company. (2) Plasmids and strains

Plasmids pPIC3, pPIC3.5K and pGAPZαA, methanolic yeast strain Pichia pastorisGS115 (his4Mut ⁺ ) are all products of Invitrogen Company, and plasmid pUC19 and E. Coli DH5αF' strain are preserved in the inventor's laboratory.

Cloning of embodiment 1 preproHSA cDNA

Take 2 grams of human embryonic liver cells, and extract total RNA from human embryonic liver cells according to the method recommended by the Trizol RNA extraction kit. According to the known natural HSA gene 5' and 3' end sequences, design primers as follows:

Primer 1:

Primer 2:

Human serum albumin preproHSA cDNA was synthesized by PCR reverse transcription using the extracted total RNA of human embryonic liver cells as a template. The reaction conditions were: denaturation at 94°C for 45 seconds, annealing at 55°C for 1 minute, extension at 72°C for 1 minute and 30 seconds, a total of 40 cycles, and finally 10 minutes at 72°C. After the PCR product was preliminarily confirmed by 1% agarose electrophoresis, the PCR product was blunted with Klenow fragment polymerase according to the recommended method of using Klenow polymerase from GIBCO BRL Company, and the blunted PCR amplified fragment was recovered using the Promega DNA recovery kit.

Ligate the filled-in PCR product with the PUC19 vector cut by SmaI. The ligation reaction system is: PUC19 (cut by SmaI) 1 μl, 5× ligation buffer 3 μl, T4 DNA ligase (1U/μl) 1.5 μl, SmaI (1U /μl) 0.5 μl, PCR fill-in product 5 μl, sterile water 4 μl. The above mixture was connected at 21°C for 5 hours. The resulting ligation product was transformed into E.coli DH5αF' competent cells, and the ampicillin plate was applied to screen positive clones. Plasmid DNA was prepared using Promega plasmid extraction kit. The resulting plasmid DNA was digested with BamHI and EcoRI to preliminarily identify the recombinant plasmid pUC19-HSA. DNA sequence analysis showed (Figure 1): The Chinese HSA gene amplified by PCR was basically consistent with that reported in the literature, and only Arg (AGA) at position 122 of the preproHSA polypeptide sequence was changed to Ser (AGT) and Glu (GAA) at position 466 Change to Gly (GGA), 162 change (TTA to CTA) does not cause amino acid changes.

Example 2 Construction of HSA Yeast Recombinant Expression Plasmids pPIC3.5K-HSA and pPIC3-HSA

The preproHSA gene fragment in the PUC19-HSA cloning vector was excised with BamHI and EcoRI double enzymes, electrophoresed on 1% agarose, and a preproHSA gene fragment of about 1.8Kb was recovered by Promega PCR Prep Kit. The recovered fragment was ligated with the pPIC3.5K expression plasmid digested with the same enzyme, the ligation reaction system was: pPIC3.5K (BamHI, EcoRI double cut) 1 μl, 5× ligation buffer 3 μl, T4 DNA ligase (1U/μl) 1.5 μl , preproHSA 3μl, sterile water 6.5μl. The above mixture was connected at 21°C for 5 hours. The resulting ligation product was transformed into E.coli DH5αF' competent cells, and the ampicillin plate was applied to screen positive clones. Promega Plasmid DNA Extraction Kit to prepare plasmid DNA. The resulting plasmid DNA was digested with BamHI and EcoRI to obtain the recombinant expression plasmid pPIC3.5K-HSA ( FIG. 2 ).

In the same way, the recombinant expression plasmid pPIC3.5K-HSA constructed above was digested by SacI and SalI, and the HSA gene fragment was recovered, and connected with the yeast expression plasmid pPIC3 digested with the same enzyme, and the recombinant expression plasmid of yeast HSA could be obtained by screening for enzyme digestion verification. pPIC3-HSA (FIG. 3).

Example 3 Construction of HSA Yeast Constitutive Recombination Expression Plasmid pGAP-HSA

The constructed pPIC3.5K-HSA recombinant expression plasmid DNA was digested with BamHI, and then treated with nuclease S1 at 23°C for 15 minutes to remove the sticky ends produced by BamHI digestion, and the linear DNA was recovered with the Promega DNA Recovery Kit. The resulting blunt-ended linear DNA was digested with EcoRI, electrophoresed on 1.5% low-melting point agarose, and a preproHSA gene fragment of about 1.8 Kb was recovered from the gel. After the pGAPZαA expression plasmid was digested with NspV, the cohesive ends were removed with nuclease S1, EcoRI digestion, 1% low melting point agarose electrophoresis and the 2.9Kb fragment was recovered in the same manner as above. The obtained linearized DNA fragment was ligated with the preproHSA gene fragment derived from pPIC3.5-HSA, and the system was: pGAPZαA (NspV+S1+EcoRI) 2 μl, preproHSA (BamHI+S1+EcoRI) 8 μl, 5× ligation buffer 3μl, T4 DNA ligase (1U/μl) 2μl. The above mixture was ligated overnight at 16°C. The resulting ligation product was transformed into E.coli DH5αF' competent cells, and spread on SLB/Zeocin plates (0.5% yeast extract, 1% peptone, 0.5% NaCl, 25mg/l Zeocin) to screen for positive clones. Promega Plasmid DNA Extraction Kit to prepare plasmid DNA. The resulting plasmid DNA was digested with BglII and EcoRI to identify the constitutive recombinant expression plasmid pGAP-HSA (Fig. 4).

Example 4 pPIC3.5k-HSA electrotransformation of yeast cells and its expression

About 10 μg of the constructed recombinant expression plasmid pPIC3.5K-HSA was digested and linearized with SalⅠ, and the linear DNA was recovered by ethanol precipitation and dissolved in 10 μl sterile water. At the same time, the empty pPIC3.5K plasmid was digested and linearized with the same enzyme and recovered as a control . Inoculate GS115 in 500ml YPD medium (1% yeast extract, 2% peptone, 2% glucose), culture at 28-30°C until _OD600 is 1.3-1.5, centrifuge at 5000r/min for 5min, and use 100ml ice-cold sterile Water, 20ml of ice-cold sterile water and 20ml of 1mol/l ice-cold sorbitol were washed once respectively. After each wash, they were centrifuged at 5000r/min for 5min to collect the cells, and finally the centrifuged cells were suspended in 200μl of 1M ice-cold sorbitol to obtain Shock competent cells. The above-mentioned linearized DNA was mixed with 80 μl GS115 electroporation cells respectively, and electroporated by GIBCOBRL electrotransformer CELL-PORATOR. The electroporation conditions were: voltage 1500V, capacitance 50 μF, resistance 4KΩ. Transfer the electroporation products to a sterile microcentrifuge tube, immediately add 0.50mL 1mol/l sorbitol pre-cooled in an ice bath, let it stand at 30 for one hour, add 0.5ml YPD, incubate at 30°C and 200rpm for 1-1.5 hours, and centrifuge quickly Remove the supernatant, add 300 μl of sterile water to suspend the bacteria, take 100 μl each, and coat the YPD plate containing different concentrations of G418 (containing 0.5 mg/ml, 1.0 mg/ml and 1.5 mg/ml of G418), 30 ° C for 2 to 4 Two days later, the positive clones were spotted on MD plates (13.4g/l YNB, 4×10 ^-4 g/l biotin, 20g/l glucose, 20g/l agar) to verify his+ phenotype, and the positive clones after 2 days at 30°C were Pichia pastoris GS115/HSA (his+Mut+) recombinant clones, the positive clones on the YPD plate containing high concentration of G418 are multi-copy recombinant clones.

A HSA-producing engineering strain (GS115/pPIC3.5k-HSA) obtained by the above construction and screening was inoculated into a 3ml YPD test tube, cultivated overnight at 30°C in a 300r/min shaker, and inoculated with 0.1-0.2% containing 8ml BMGY (10g/l yeast cream, 20g/l peptone, 0.1mol/l potassium phosphate buffer pH6.0, 13.4g/l YNB, 4×10 ^-4 g/l biotin, 10g/l glycerol) in a 50ml culture tube. Cultivate at 30°C and 300r/min until the OD ₆₀₀ is 2-10. Centrifuge at 5000r/min for 5-10min at room temperature. The collected bacteria were suspended with 40ml of BMMY (10g/l glycerol in BMGY was changed to 5ml/l methanol) and then transferred to a 250ml Erlenmeyer flask, and induced at 28°C and 300r/min. Add methanol to 5ml/l every 24 hours. After 48 hours of induction, the supernatant of the fermentation broth was detected by self-made HSA antiserum ELISA, and the expression level of HSA was about 60-80 mg/l.

Example 5 Obtaining of Glucose Non-inhibiting HSA Engineering Bacteria GS115/pPIC3-HSA ⁺

The HSA recombinant expression plasmid pPIC3-HSA obtained in Example 2 was electrotransformed into yeast cell GS115 according to the steps described in Example 4, and then spread on the MD plate, and after cultivating at 30°C for 2-4 days, positive clones were passed through the MM plate (13.4g/l YNB, 4×10 ^-4 g/l biotin, 5ml/l methanol, 20g/l agar) were verified, and a methanol utilization normal HSA expression clone (GS115/pPIC3-HSA) was selected.

The obtained HSA expression clone GS115/pPIC3-HSA was inoculated into a 3ml YPD test tube and cultured overnight at 28°C and 300r/min. Take 0.6ml of bacterial liquid and centrifuge at 15000r/min for 1min at room temperature. The bacteria were collected, washed repeatedly with sterile water, and then resuspended in 2 ml of sodium phosphate buffer (pH 7.0). Add 60 μl EMS stock solution, mix evenly, and incubate at 28° C. on a rotary shaker for 1 hr. _Inactivate EMS by treating _with 400 volumes of sterile 5% _Na2S2O3 . Centrifuge at 15000r/min at room temperature for 1min, collect the bacteria, and suspend in 2ml sterile water. Coat bm-YNB plates containing 0.02% 2-deoxyglucose at different concentrations (0.1mol/l phosphate buffer pH6.0, 5ml/l methanol, 13.4g/lYNB, 15g/l agar), and culture at 30°C for 2 -5 days. The obtained positive clones were inoculated into 3ml YPD test tubes and cultured overnight at 28-30°C and 300r/min. 1% was transferred to 10ml YPDM (10g/l yeast extract, 20g/l peptone, 20g/l glucose, 5ml/l methanol), induced at 28-30°C, 300r/min for 2-3 days, and methanol was added every day until the end. The concentration is 5ml/l. SDS-PAGE electrophoresis of the supernatant of the fermentation broth showed that the EMS mutagenized strain (GS115/pPIC3-HSA ⁺ ) had an obvious 67kD protein band than the unmutated strain (GS115/pPIC3-HSA) ( FIG. 5 ).

Embodiment 6 Contains two kinds of promoter multiple expression cassettes HSA engineering bacterium GS115/pPIC3.5k+p GAP

About 10 μg of the recombinant expression plasmid pGAP-HSA constructed in Example 3 was digested with PshAI and linearized, and the linear DNA was recovered by ethanol precipitation and dissolved in 10 μl sterile water. The same experimental procedure as in Example 4 was used to prepare GS115/pPIC3.5k-HSA electroshock competent cells. The above-mentioned linearized DNA was mixed with 80 μl of electroporated cells respectively, and the GIBCO BRL electroporation instrument CELL-PORATOR was used for electroporation transformation according to the electroporation transformation step in Example 4. Finally, take 100 μl of transformed cells and spread them on YPDS plates containing different concentrations of Zeocin (10g/l yeast extract, 20g/l peptone, 20g/l glucose, 1mol/l sorbitol, 20g/l agar, and the concentration of Zeocin is 100mg/l or 500mg /l), cultured at 30° C. for 2 to 4 days, and the positive clone obtained was an engineering bacterium containing two kinds of promoters pAOX1 and pGAP to express HSA. The positive clones on the YPDS plate containing high concentration of Zeocin are pGAP-HSA multi-copy recombinant clones. Through the above steps, a HSA engineered strain containing two kinds of promoter multi-expression cassettes GS115/pPIC3.5k+p GAP was finally screened. This strain can express HSA in medium YPD or BMGY without methanol, indicating constitutive recombination The expression plasmid pGAP-HSA can express HSA in yeast. As determined by ELBA analysis, the HSA expression level of this strain can reach about 120 mg/l after 2 days of methanol induction.

Example 7 Construction of HSA Engineering Strain Pichia pastoris SIB 121 Containing Two Kinds of Promoter Multiple Expression Cassettes

In the same manner as in Example 6, the HSA strain GS115/pPIC3-HSA ⁺ obtained in Example 5 was used as the host cell, electrotransformed into the constitutive recombinant expression plasmid pGAP-HSA, and induced and screened to obtain an engineering strain GS115/pAOX1+pGAP (Pichia pastoris SIB 121). This engineering bacteria can be prepared in YPDM or BMMGY (10g/l yeast extract, 20g/L peptone, 0.1mol/l potassium phosphate buffer pH6.0, 13.4g/lYNB, 4×10 ^-4 g/l biotin, 10g/l 1 glycerol, 5ml/l methyl alcohol) medium simultaneously start two promotors to express HSA, shake flask fermented liquid shows through SDS-PAGE analysis, and its expression amount is obviously higher than the engineering bacterium of single promotor (Fig. 6, Fig. 7) . ELISA analysis with self-made HSA antiserum showed that the HSA expression level of SIB 121 could reach nearly 120 mg/l (shake flask) after two days 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.

Images