WO2017190671A1 - 高效表达重组人血清白蛋白工程菌的构建 - Google Patents

高效表达重组人血清白蛋白工程菌的构建 Download PDF

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WO2017190671A1
WO2017190671A1 PCT/CN2017/083079 CN2017083079W WO2017190671A1 WO 2017190671 A1 WO2017190671 A1 WO 2017190671A1 CN 2017083079 W CN2017083079 W CN 2017083079W WO 2017190671 A1 WO2017190671 A1 WO 2017190671A1
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rhsa
expression
yeast
pdi
serum albumin
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PCT/CN2017/083079
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French (fr)
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常国栋
窦鑫
熊小波
王文超
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北京普罗吉生物科技发展有限公司
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Priority to JP2019510736A priority Critical patent/JP7014774B2/ja
Priority to US16/099,159 priority patent/US20190330707A1/en
Priority to EP17792497.4A priority patent/EP3453763B1/en
Priority to AU2017259656A priority patent/AU2017259656B2/en
Priority to CN201780027907.9A priority patent/CN109415735B/zh
Priority to CA3023046A priority patent/CA3023046A1/en
Publication of WO2017190671A1 publication Critical patent/WO2017190671A1/zh
Priority to IL262752A priority patent/IL262752B/en
Priority to JP2022005006A priority patent/JP7448977B2/ja

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Definitions

  • the present invention relates to recombinant production of human serum albumin, in particular, the present invention relates to a method for efficiently producing human serum albumin by co-expressing human serum albumin and one or more human serum albumin expression promoting factors in yeast cells. .
  • HSA Human Serum Albumin
  • rHSA Human serum albumin recombinantly produced by bioengineering technology
  • yeast expression rHSA technology is the most extensive and mature.
  • Patent US 5,683,893 discloses a method for mutating a promoter of the Alcohol oxidase (AOX) promoter, which enhances the expression of rHSA in yeast.
  • AOX Alcohol oxidase
  • Chinese Patent Application No. 200510068171.9 filed on Apr. 29, 2005, discloses a rHSA yeast strain construction and fermentation method, and the expression amount can reach 10 g/L medium supernatant.
  • the above methods still have the defects of low expression of rHSA, long fermentation time and low production efficiency, and it is necessary to find a new method to construct a more productive engineering strain.
  • Pichia has a post-translational modification function of eukaryotic proteins, so that foreign proteins can be correctly folded, assembled and secreted extracellularly after expression.
  • Pichia pastoris can effectively use methanol as a single carbon source for high-density fermentation. Therefore, Pichia pastoris has been widely used for the expression of foreign proteins.
  • Pichia pastoris generally has a long fermentation cycle, high production cost, and is prone to contamination and protein degradation. Therefore, shortening the fermentation time and reducing the cost has become a research hotspot of the expression system.
  • Endoplasmic reticulum is an important site for protein folding into natural conformation and post-modification such as glycosylation and phosphorylation.
  • unfolded protein response UPR
  • UPR unfolded protein response
  • Transcriptional activator HAC1 acts as a regulator of yeast UPR and regulates the expression of a series of proteins related to UPR, including binding protein KAR2 (Binding protein KAR2) and protein disulfide-isomerase (Protein disulfide-isomerase). , PDI), endoplasmic reticulum oxidoreductin-1 (ERO1), Peptidyl-prolyl cis-trans isomerase (PPI), etc., which help to express and secrete proteins of interest. The process plays an important role.
  • 201310095971.4 discloses a method for co-expression of PDI with Aspergillus niger alpha-glucosidase, which increases the expression level of the protein of interest.
  • Chinese Patent Application No. 200780026864.9 filed on May 16, 2007, discloses a method for enhancing the expression of HAC1 in methanol assimilation yeast (Ogataea minuta), and the obtained engineering strain has a high protein secretion ability.
  • Tiziana Lodi et al. reported that ERO1 contributes to the secretion of rHSA in Kluyveromyces lactis (Tiziana Lodi. et al. AEM 2005; 71: 4359-4363).
  • KAR2 has doubled the expression level of the human single-chain antibody fragment (A33scFv) (Leonardo M. Damasceno, et al. Appl Microbiol Biotechnol, 2007; 74:381-389).
  • the present invention provides a method for efficiently expressing recombinant human serum albumin, which comprises the steps of: (a) human serum albumin gene and (b) expression of one or more rHSA in a yeast host cell. Factor genes are co-expressed.
  • the invention also provides an engineered bacteria for efficiently expressing recombinant human serum albumin, wherein the engineered bacteria is yeast, and comprises: (a) a human serum albumin gene and (b) one or more rHSA expression promoting factor genes. .
  • the yeast is Pichia; preferably, the yeast is Pichia pastoris.
  • the rHSA expression promoting factor is selected from the group consisting of a transcriptional activator HAC1, a binding protein KAR2, a protein disulfide isomerase (PDI), an endoplasmic reticulum oxidoreductase (ERO1), and a proline A group of enzymes (PPI).
  • a transcriptional activator HAC1 a binding protein KAR2
  • PDI protein disulfide isomerase
  • ERO1 endoplasmic reticulum oxidoreductase
  • PPI proline A group of enzymes
  • the following combinations are co-expressed in the yeast host cell:
  • the human serum albumin gene of the invention can be transformed into the yeast host cell by a plasmid; the rHSA expression promoting factor gene can be transformed into the yeast host by one, two or more plasmids cell.
  • the engineered bacteria of the present invention need not inactivate the original rHSA expression promoting factor gene in the host genome, and thus the obtained engineered bacteria can simultaneously contain the transferred HSA gene and the rHSA expression promoting factor gene and the host cell.
  • the original rHSA expression promoting factor gene is not inactivated.
  • the engineered bacteria of the present invention can express rHSA efficiently, wherein the expression level of rHSA in the co-expressing strain is significantly increased, and the fermentation supernatant can be up to 18.2 g/L, which lays a foundation for large-scale industrial production of rHSA. Solid foundation.
  • Figure 1 shows the DNA sequence encoding HSA.
  • Figure 2 shows the amino acid sequence of HSA encoded by the DNA sequence shown in Figure 1.
  • Figure 3 shows the DNA sequence encoding Pichia pastoris ERO1.
  • Figure 4 shows the amino acid sequence encoding ERO1 from the DNA sequence shown in Figure 3.
  • Figure 5 shows the DNA sequence encoding Pichia pastoris HAC1.
  • Figure 6 shows the amino acid sequence encoding HAC1 from the DNA sequence shown in Figure 5.
  • Figure 7 shows the DNA sequence encoding Pichia pastoris PDI.
  • Figure 8 shows the amino acid sequence encoding the PDI from the DNA sequence shown in Figure 7.
  • Figure 9 shows the DNA sequence encoding the Pichia pastoris PPI.
  • Figure 10 shows the amino acid sequence encoding the PPI by the DNA sequence shown in Figure 9.
  • Figure 11 shows the DNA sequence encoding Pichia pastoris KAR2.
  • Figure 12 shows the amino acid sequence encoding KAR2 from the DNA sequence shown in Figure 11.
  • Figure 13 shows the rHSA Pichia pastoris secretion expression vector.
  • Figure 14 shows the pPICZ ⁇ -ERO1 Pichia expression vector
  • Figure 15 shows the pPIC6-HAC1 Pichia pastoris expression vector
  • Figure 16 shows the pPICZ ⁇ -PDI Pichia expression vector
  • Figure 17 shows the pPIC6-PPI Pichia expression vector
  • Figure 18 shows the pPIC6-KAR2 Pichia expression vector
  • Figure 19 shows the results of shake flask expression electrophoresis of the rHSA co-expressing strain.
  • rHSA expression promoting factor refers to various protein factors capable of promoting the expression of rHSA, the source of which is not limited to a particular species. Specifically, a protein having molecular chaperone activity, such as KAR2; a folding enzyme such as PDI; and a transcriptional regulator such as HAC1 and the like are included.
  • Specific rHSA expression promoting factors particularly suitable for the present invention include: transcriptional activator HAC1, binding protein KAR2, protein disulfide isomerase (PDI), endoplasmic reticulum oxidoreductase (ERO1) and proline Structured enzyme (PPI) and the like.
  • rHSA expression promoting factor is not limited to a particular species.
  • an rHSA expression promoting factor derived from Saccharomyces cerevisiae, such as PDI can function well in Pichia pastoris.
  • rHSA expression promoting factor also includes substitutions, additions or deletions having one or several amino acid residues in amino acid sequence compared to the above expression promoting factors, and having substantially similar biological functions. Proteins or active fragments may also include modified products, fusion proteins and complexes containing these proteins or active fragments thereof.
  • the rHSA expression promoting factor is derived from the host cell.
  • the rHSA expression promoting factor from Pichia pastoris is preferably introduced into Pichia host cells for expression.
  • the rHSA expression promoting factor may be introduced alone or in combination.
  • a rHSA expression promoting factor (including ERO1, HAC1, KAR2, PDI, PPI, etc.) is introduced into a host cell alone, co-expressed with rHSA, and significantly increased in expression.
  • PDI is co-expressed with rHSA compared to the expression level when no expression promoting factor is used, resulting in an increase in the expression level of rHSA by 160%.
  • the rHSA expression promoting factor can be introduced into a host cell in combination.
  • PDI and HAC1 are used in combination compared to the expression level when no expression promoting factor is used, so that the expression level of rHSA is nearly doubled.
  • three or more rHSA expression promoting factors can be introduced into a host cell.
  • rHSA is co-expressed with three expression promoting factors PDI, PPI and HAC1 in a host cell, significantly increasing the expression level of rHSA.
  • the inventors cloned the ERO1, HAC1, KAR2, PDI, and PPI genes of Pichia pastoris GS115 strain by genetic engineering techniques, and constructed an inducible expression vector by co-expression of these proteins with rHSA. Through various combinations of screening, high-yield, high-efficiency yeast engineering bacteria were obtained.
  • the expression vector pPIC9K (purchased from Invitrogen) carries a yeast alpha-factor signal peptide that can be used to secrete and express foreign proteins.
  • the following primers were designed according to the sequence of NM_000477.5 published by GenBank: (the enzyme cleavage site is underlined)
  • HSA Reverse CCG GAATTC TTATAAGCCTAAGGCAGCTTGACTTGC (SEQ ID NO: 2)
  • the human liver cDNA library was used as a template to carry out polymerase chain reaction (PCR) under specific conditions: denaturation at 94 ° C for 3 minutes; denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes, a total of 30 Cycle; then extend at 72 ° C for 10 minutes.
  • the obtained PCR product was digested with Xho I and EcoR I, and inserted into the pPIC9K vector to obtain the vector pPIC9K-HSA, and the structure is shown in FIG.
  • the HSA DNA sequence was sequenced correctly and the results are shown in Figure 1.
  • the corresponding amino acid sequence is shown in Figure 2.
  • Pichia pastoris GS115 (purchased from Invitrogen) is used as a host strain, and the pPIC9K-HSA vector is linearized by Sal I digestion and electroporated into GS115 strain.
  • Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ).
  • the insert was integrated into the HIS4 locus of GS115 chromosome, and the transformed strain was subjected to antibiotic enrichment screening using YPD (Yeast extract Peptone Dextrose Medium) solid medium containing 2 mg/mL geneticin (G418) to obtain a yeast strain capable of secreting rHSA. GS115-rHSA.
  • the DNA sequence of the Pichia pastoris ERO1 gene was obtained from the NCBI database, and the following primers were designed for gene amplification: (the enzyme cleavage site is underlined)
  • the ERO1 gene was obtained by PCR, digested with SnaB I and Not I, and inserted into the expression vector pPICZ ⁇ (purchased from Invitrogen) to obtain the vector pPICZ ⁇ -ERO1.
  • the structure is shown in Figure 14. Show.
  • the ERO1 DNA sequence was sequenced correctly, as shown in Figure 3.
  • the corresponding amino acid sequence is shown in Figure 4.
  • the DNA sequence of the Pichia pastoris HAC1 gene was obtained from the NCBI database, and the following primers were designed for gene amplification: (the enzyme cleavage site is underlined)
  • HAC Forward CGG TTCGAA ACGATGCCCGTAGATTCTTCT (SEQ ID NO: 5)
  • the DNA sequence of the Pichia pastoris PDI gene was obtained from the NCBI database, and the following primers were designed for gene amplification: (the enzyme cleavage site is underlined)
  • the PDI gene was obtained by PCR, digested with SnaB I and Not I, and inserted into the expression vector pPICZ ⁇ (purchased from Invitrogen) to obtain the vector pPICZ ⁇ -PDI.
  • the structure is shown in Figure 16. Show.
  • the PDI DNA sequence was sequenced correctly and the results are shown in Figure 7.
  • the corresponding amino acid sequence is shown in Figure 8.
  • the DNA sequence of the PPI gene of Pichia pastoris was obtained from the NCBI database, and the following primers were designed for gene amplification: (the enzyme cleavage site is underlined)
  • the PPI gene was obtained by PCR using the genome of Pichia pastoris GS115 as a template, and digested with SnaB I and Not I, and inserted into the expression vector pPIC6 (purchased from Invitrogen) to obtain the vector pPIC6-PPI. Shown.
  • the DNA sequence was sequenced correctly, the sequence is shown in Figure 9, and the corresponding amino acid sequence is shown in Figure 10.
  • the DNA sequence of the Pichia pastoris KAR2 gene was obtained from the NCBI database, and the following primers were designed for gene amplification: (the enzyme cleavage site is underlined)
  • KAR2Forward CGG TTCGAA ACGATGCTGTCGTTAAAACCATCT (SEQ ID NO: 11)
  • KAR2Reverse GCTG GCGGCCGC CTATGATCATGATGAGTTGTAG (SEQ ID NO: 12)
  • the KAR2 gene was obtained by PCR, digested with SnaB I and Not I, and inserted into the expression vector pPIC6 (purchased from Invitrogen) to obtain the vector pPIC6-KAR2. Shown.
  • the DNA sequence was sequenced correctly, the sequence is shown in Figure 11, and the corresponding amino acid sequence is shown in Figure 12.
  • the rHSA secreting expression strain GS115-rHSA was used as the original strain, and the above constructed pPICZ ⁇ -ERO1 vector was linearized by Sac I and electrotransformed into GS115-rHSA strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 2 mg/mL zeamycin (Zeocin), and yeast strain GS115-rHSA-ERO1 co-expressing ERO1 and rHSA was obtained.
  • Zeocin zeamycin
  • the rHSA secretory expression strain GS115-rHSA was used as the original strain, and the pPIC6-HAC1 vector constructed in Example 4 was linearized by Sac I and electrotransformed into GS115-rHSA strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 1 mg/mL of blasticidin, and yeast strain GS115-rHSA-HAC1 co-expressing HAC1 and rHSA was obtained.
  • the rHSA secreted expression strain GS115-rHSA was used as the original strain, and the above constructed pPICZ ⁇ -PDI vector was linearized by Sac I and electrotransformed into GS115-rHSA strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 2 mg/mL zeamycin (Zeocin), and yeast strain GS115-rHSA-PDI co-expressing PDI and rHSA was obtained.
  • Zeocin zeamycin
  • the rHSA secreted expression strain GS115-rHSA was used as the original strain, and the pPIC6-PPI vector constructed in Example 6 was linearized by Pme I digestion and electroporated into GS115-rHSA strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 1 mg/mL of blasticidin, and a yeast strain GS115-rHSA-PPI co-expressed with PPI and rHSA was obtained.
  • the pHS6-KHS2 vector constructed in Example 7 was linearized by Pme I and electroporated into GS115-rHSA strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 1 mg/mL of blasticidin, and a yeast strain GS115-rHSA-KAR2 co-expressed with KAR2 and rHSA was obtained.
  • the expression strain GS115-rHSA-PDI was used as the original strain, and the above constructed pPIC6-HAC1 vector was linearized by Sac I digestion and electroporated into GS115-rHSA-PDI strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA-PDI strain. The transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 1 mg/mL of blasticidin, and yeast strain GS115-rHSA-PDI-HAC1 co-expressed with HAC1, PDI and rHSA was obtained.
  • the expression strain GS115-rHSA-PDI screened in Example 10 was the original strain, and the pPIC6-PPI vector constructed in Example 6 was linearized by Pme I digestion and electroporated into the GS115-rHSA-PDI strain. Methods for the preparation of competent and electroporation are referred to the literature (James M. Cregg Pichia Protocols 2 nd ). The insert was integrated into the chromosome 5'AOX site of the GS115-rHSA-PDI strain.
  • the transformed strain was subjected to antibiotic enrichment screening using YPD solid medium containing 1 mg/mL of blasticidin, and a yeast strain GS115-rHSA-PDI-PPI co-expressed with PPI, PDI and rHSA was obtained.
  • GS115-rHSA-ERO1, GS115-rHSA-HAC1, GS115-rHSA-PDI, GS115-rHSA-PPI, GS115-rHSA-KAR2, GS115-rHSA-PDI-HAC1 and GS115-rHSA were screened in the above examples.
  • -PDI-PPI strain single colony inoculated into 2 ml of MGY (1.34% yeast nitrogen source base; 1.0% glycerol; 4.0 ⁇ 10 -5 biotin) medium, cultured at 30 ° C for 16 hours, collected by centrifugation, transferred to 20 ml of BMMY (1.0% yeast extract; 2.0% peptone; 0.1 M potassium phosphate buffer, pH 6.0; 1.34% yeast nitrogen source base; 0.5% anhydrous methanol) medium culture, induced expression for 72 hours, of which every 12 Add 50 ⁇ l of anhydrous methanol to the hour. After the end of the induction, the culture supernatant was taken for SDS-PAGE electrophoresis (Fig. 19). Compared with the control strain (GS115-rHSA), the expression levels of 7 co-expressing strains rHSA were increased, and the expression rate was as shown in Table 1 using Quantity One software.
  • the PDI-HAC1 and GS115-rHSA-PDI-PPI strains were fermented, and the fermentation conditions were referred to the Pichia Fermentation Process Guidelines published by Invitrogen.
  • the fermentation was terminated 80 hours after the induction of expression, and the culture supernatant was taken to analyze the expression level of rHSA.
  • Table 2 When the fixed fermentation time was 80 hours, the expression level of the co-expressing strain rHSA was significantly increased, and the highest fermentation supernatant was 18.2 g/L, which laid a foundation for large-scale industrial production of rHSA.

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Abstract

本发明提供了一种高效表达重组人血清白蛋白的方法，其特征在于包括以下步骤：在酵母宿主细胞中，将(a)人血清白蛋白基因与(b)一种或多种rHSA表达促进因子基因进行共表达。

Description

高效表达重组人血清白蛋白工程菌的构建 技术领域
本发明涉及人血清白蛋白的重组生产，具体而言，本发明涉及通过在酵母细胞中共表达人血清白蛋白与一种或多种人血清白蛋白表达促进因子来高效生产人血清白蛋白的方法。
背景技术
人血清白蛋白(Human Serum Albumin，HSA)是人血中含量最丰富的蛋白质，占血浆总蛋白含量约60％，具有重要的生理功能，可维持血液渗透压，是运输内源性及外源性物质的重要载体和重要的血液缓冲组分。此外，HSA还可以作为细胞培养基的添加成分、药物辅料等，具有重要应用价值。目前，HSA的来源主要有两种：一种是从血浆中提取，由于国内血浆紧缺以及存在艾滋病、肝炎等病毒感染的风险，该方法获得的HSA无法满足巨大的市场需求；另一种是利用生物工程技术进行重组制备。利用生物工程技术重组制备的人血清白蛋白叫做重组人血清白蛋白(Recombinant Human Serum Albumin，rHSA)。其中，酵母表达rHSA技术研究得最广泛、最成熟。专利US 5683893公开了一种对毕赤酵母醇氧化酶(Alcohol oxidase，AOX)启动子进行突变的方法，增强了rHSA在酵母中的表达。2005年4月29日提交的中国专利申请200510068171.9公开了一种rHSA酵母菌株构建及发酵方法，表达量可以达到10g/L培养基上清。但是，上述方法仍然存在rHSA表达量较低、发酵时间长、生产效率低等缺陷，需要寻找新的方法构建更加高产的工程菌株。
毕赤酵母(Pichia)具有真核生物蛋白翻译后修饰功能，使得外源蛋白在表达后能够正确折叠、组装并分泌到胞外。同时，毕赤酵母能有效利用甲醇作为单一碳源进行高密度发酵。因此，毕赤酵母已经广泛用于外源蛋白的表达。但是，毕赤酵母的发酵周期一般较长、生产成本高，并且容易发生污染及蛋白降解。所以，缩短发酵时间、降低成本成为该表达体系的研究热点。
酵母内质网(Endoplasmic reticulum，ER)是蛋白质折叠成天然构象、进行糖基化和磷酸化等后修饰的重要场所。当内质网中存在大量未折叠蛋白时，会诱发未折叠蛋白应答效应(unfolded protein response，UPR)，进而激活下游分子伴侣、折叠酶的表达以及内质网相关的蛋白质降解途径。作为自身调节机制，UPR对酵母生长及分泌蛋白的表达具有重要的作用(Graham Whyteside,et al.FEBS Letters 2011；585:1037-1041)。转录激活因子HAC1(Transcriptional activator HAC1)作为酵母UPR的调控因子，能够调控与UPR相关的一系列蛋白质的表达，包括结合蛋白KAR2(Binding protein KAR2)、蛋白质二硫键异构酶(Protein disulfide-isomerase，PDI)、内质网氧化还原酶(Endoplasmic reticulum oxidoreductin-1，ERO1)、脯氨酸顺反异构酶(Peptidyl-prolyl cis-trans isomerase，PPI)等，它们在帮助目的蛋白质表达与分泌的过程中发挥着重要的作用。2013年3月22日提交的中国专利申请201310095971.4公开了一种将PDI与黑曲霉α-葡萄糖苷酶共表达的方法，增加了目的蛋白的表达量。2007年5月16日提交的中国专利申请200780026864.9公开了一种增强甲醇同化酵母(Ogataea minuta)HAC1表达的方法，获得的工程菌株有较高的蛋白分泌能力。Tiziana Lodi等报道在乳酸克鲁维酵母(Kluyveromyces lactis)中，ERO1能有助于rHSA的分泌(Tiziana Lodi.et al.AEM 2005；71:4359-4363)。此外，在毕赤酵母中共表达KAR2使人源单链抗体片段(A33scFv)的表达量提高了两倍(Leonardo M.Damasceno,et al.Appl Microbiol Biotechnol,2007；74:381-389)。
发明内容
本发明提供了一种高效表达重组人血清白蛋白的方法，其特征在于包括以下步骤：在酵母宿主细胞中，将(a)人血清白蛋白基因与(b)一种或多种rHSA表达促进因子基因进行共表达。
将外源的人血清白蛋白基因与rHSA表达促进因子基因引入酵母宿主细胞后，显著提高了rHSA的表达量。
本发明还提供了一种高效表达重组人血清白蛋白的工程菌，其中该工程菌为酵母，并且包含：(a)人血清白蛋白基因与(b)一种或多种rHSA表达促进因子基因。
在一些实施方案中，其中所述酵母为毕赤酵母属(Pichia)；优选地，所述酵母为巴斯德毕赤酵母(Pichia pastoris)。
在一些实施方案中，所述rHSA表达促进因子选自由转录激活因子HAC1、结合蛋白KAR2、蛋白质二硫键异构酶(PDI)、内质网氧化还原酶(ERO1)和脯氨酸顺反异构酶(PPI)构成的组。
在本发明的一些实施例中，所述酵母宿主细胞中共表达以下组合：
rHSA与ERO1；
rHSA与PDI；
rHSA、PDI与HAC1；
rHSA、PPI与KAR2；或者
rHSA、PDI、PPI与HAC1。
在一些实施方案中，本发明的人血清白蛋白基因可以通过质粒转化入所述酵母宿主细胞；所述rHSA表达促进因子基因可以通过1个、2个或更多个质粒转化入所述酵母宿主细胞。
在一些实施方案中，本发明的工程菌中无需使宿主基因组中原有的rHSA表达促进因子基因失活，因此所获得的工程菌可以同时包含转入的HSA基因和rHSA表达促进因子基因以及宿主细胞中原有的rHSA表达促进因子基因。
在本发明的一些实施方案中，本发明的工程菌可以高效表达rHSA，其中共表达菌株中rHSA的表达量明显提高，最高可达18.2g/L发酵上清，为rHSA大规模工业化生产奠定了坚实基础。
附图说明
图1显示编码HSA的DNA序列。
图2显示由图1所示DNA序列编码HSA的氨基酸序列。
图3显示编码毕赤酵母ERO1的DNA序列。
图4显示由图3所示DNA序列编码ERO1的氨基酸序列。
图5显示编码毕赤酵母HAC1的DNA序列。
图6显示由图5所示DNA序列编码HAC1的氨基酸序列。
图7显示编码毕赤酵母PDI的DNA序列。
图8显示由图7所示DNA序列编码PDI的氨基酸序列。
图9显示编码毕赤酵母PPI的DNA序列。
图10显示由图9所示DNA序列编码PPI的氨基酸序列。
图11显示编码毕赤酵母KAR2的DNA序列。
图12显示由图11所示DNA序列编码KAR2的氨基酸序列。
图13显示了rHSA毕赤酵母分泌表达载体。
图14显示了pPICZα-ERO1毕赤酵母表达载体
图15显示了pPIC6-HAC1毕赤酵母表达载体
图16显示了pPICZα-PDI毕赤酵母表达载体
图17显示了pPIC6-PPI毕赤酵母表达载体
图18显示了pPIC6-KAR2毕赤酵母表达载体
图19显示了rHSA共表达菌株摇瓶表达电泳结果。
具体实施方式
本文所用的术语“rHSA表达促进因子”是指能够促进rHSA表达的各种蛋白因子，其来源并不局限于特定的种属。具体而言，包括具有分子伴侣活性的蛋白，例如KAR2；折叠酶，例如PDI；以及转录调节因子，例如HAC1等。
特别适合于本发明的具体的rHSA表达促进因子包括：转录激活因子HAC1、结合蛋白KAR2、蛋白质二硫键异构酶(PDI)、内质网氧化还原酶(ERO1)和脯氨酸顺反异构酶(PPI)等。
“rHSA表达促进因子”的来源并不局限于特定的种属。例如，来源于酿酒酵母的rHSA表达促进因子例如PDI，可以在毕赤酵母中很好地发挥作用。
本领域技术人员可以理解，“rHSA表达促进因子”也包括在氨基酸序列上与上述表达促进因子相比具有一个或几个氨基酸残基的取代、添加或缺失，并具有基本相似的生物学功能的蛋白质或活性片段，也可以包括含有这些蛋白质或其活性片段的修饰产物、融合蛋白及复合物。
优选地，所述rHSA表达促进因子来源于该宿主细胞。例如，来自毕赤酵母的rHSA表达促进因子优选被导入毕赤酵母宿主细胞中进行表达。
本领域技术人员可以理解，不同类促进因子的不同组合方式可以产生不同的技术效果，例如：同时加入转录调节因子HAC1和折叠酶PDI得到了比单独使用PDI更好的促进表达rHSA的效果。
所述rHSA表达促进因子可以单独引入，也可以组合引入。
例如，在本发明的一些实施方案中，一种rHSA表达促进因子(包括ERO1、HAC1、KAR2、PDI、PPI等)被单独引入宿主细胞，与rHSA共表达，并显著提高了表达量。例如，与不使用表达促进因子时的表达量相比，将PDI与rHSA共表达，使得rHSA的表达量提高了160％。
在本发明的一些实施方案中，rHSA表达促进因子可以被两两组合引入宿主细胞。例如，与不使用表达促进因子时的表达量相比，组合使用PDI和HAC1，使得rHSA的表达量提高了将近两倍。
在本发明的一些实施方案中，可以将三种或者更多种rHSA表达促进因子引入宿主细胞。例如，在本发明的具体实施方案中，rHSA与三种表达促进因子PDI、PPI与HAC1在宿主细胞中被共表达，显著提高了rHSA的表达量。
在本发明的一些实施方案中，发明人采用基因工程技术，克隆了毕赤酵母GS115菌株的ERO1、HAC1、KAR2、PDI、PPI基因，构建了诱导型表达载体，通过将这些蛋白质与rHSA共表达，经过多种组合筛选，获得了高表达、高效率的酵母工程菌。
实施例
1、HSA克隆及表达载体构建
表达载体pPIC9K(购自invitrogen公司)带有酵母α-factor信号肽，可以用于分泌表达外源蛋白。根据GenBank公布的NM_000477.5序列设计如下引物：(酶切位点以下划线标出)
HSA Forward：CCGCTCGAGAAAAGAGACGCTCACAAGAGTGAGGT(SEQ ID NO:1)
HSA Reverse：CCGGAATTCTTATAAGCCTAAGGCAGCTTGACTTGC(SEQ ID NO:2)
以人肝脏cDNA文库为模板，进行聚合酶链式反应(PCR)，具体条件为：94℃变性3分钟；94℃变性30秒，55℃退火30秒，72℃延伸2分钟，共进行30个循环；然后72℃延伸10分钟。获得的PCR产物经过Xho Ⅰ与EcoR Ⅰ酶切消化后，插入到pPIC9K载体，获得载体pPIC9K-HSA，结构如图13所示。HSA DNA序列测序正确，结果如图1所示。所对应的氨基酸序列如图2所示。
2、rHSA酵母分泌表达菌株的构建与筛选
本发明采用毕赤酵母GS115(购自invitrogen公司)作为宿主菌，pPIC9K-HSA载体经过Sal Ⅰ酶切线性化，电转化入GS115菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115染色体HIS4位点，转化菌株采用含有2mg/mL遗传霉素(G418)的YPD(Yeast extract Peptone Dextrose Medium)固体培养基进行抗生素富集筛选，获得了可以分泌表达rHSA的酵母菌株GS115-rHSA。
3、毕赤酵母ERO1基因的克隆及载体构建
从NCBI数据库中获得毕赤酵母ERO1基因的DNA序列，设计如下引物进行基因扩增：(酶切位点以下划线标出)
ERO Forward:CGGTTCGAAAGCATGAACCCTCAAATCCCTTT(SEQ ID NO:3)
ERO Reverse:GCTGGCGGCCGCTTACAAGTCTACTCTATATGTGG(SEQ ID NO:4)
以毕赤酵母GS115菌株基因组为模板，通过PCR反应获得ERO1基因，使用SnaB Ⅰ与Not Ⅰ双酶切消化，插入表达载体pPICZα(购自invitrogen公司)，获得载体pPICZα-ERO1，结构如图14所示。ERO1DNA序列测序正确，如图3所示。所对应的氨基酸序列如图4所示。
4、毕赤酵母HAC1基因的克隆及载体构建
从NCBI数据库中获得毕赤酵母HAC1基因的DNA序列，设计如下引物进行基因扩增：(酶切位点以下划线标出)
HAC Forward:CGGTTCGAAACGATGCCCGTAGATTCTTCT(SEQ ID NO:5)
HAC Reverse:GCTGGCGGCCGCCTATTCCTGGAAGAATACAAAGTC(SEQ ID NO:6)
酵母RNA提取及反转录方法参考文献(J.萨姆布鲁克等，分子克隆试验指南第三版)。以毕赤酵母GS115菌株cDNA为模板，通过PCR反应获得HAC1基因，使用SnaB Ⅰ与Not Ⅰ双酶切消化，插入表达载体pPIC6(购自invitrogen公司)，获得载体pPIC6-HAC1，结构如图15所示。HAC1DNA序列测序正确，结果如图5所示。所对应的氨基酸序列如图6所示。
5、毕赤酵母PDI基因的克隆及载体构建
从NCBI数据库中获得毕赤酵母PDI基因的DNA序列，设计如下引物进行基因扩增：(酶切位点以下划线标出)
PDI Forward:CGGTTCGAAACGATGCAATTCAACTGGAATATT(SEQ ID NO:7)
PDI Reverse:GCTGGCGGCCGCTTAAAGCTCGTCGTGAGCGTCTGC(SEQ ID NO:8)
以毕赤酵母GS115菌株基因组为模板，通过PCR反应获得PDI基因，使用SnaB Ⅰ与Not Ⅰ双酶切消化，插入表达载体pPICZα(购自invitrogen公司)，获得载体pPICZα-PDI，结构如图16所示。PDI DNA序列测序正确，结果如图7所示。所对应的氨基酸序列如图8所示。
6、毕赤酵母PPI基因的克隆及载体构建
从NCBI数据库中获得毕赤酵母PPI基因的DNA序列，设计如下引物进行基因扩增：(酶切位点以下划线标出)
PPI Forward:CGGTTCGAAACGATGGAATTAACCGCATTGCGCAGC(SEQ ID NO:9)
PPI Reverse:GCTGGCGGCCGCTTACAACTCACCGGAGTTGGTGATC(SEQ ID NO:10)
以毕赤酵母GS115菌株基因组为模板，通过PCR反应获得PPI基因，使用SnaB Ⅰ与Not Ⅰ双酶切消化，插入表达载体pPIC6(购自Invitrogen公司)，获得载体pPIC6-PPI，其结构如图17所示。DNA序列测序正确，序列如图9所示，所对应的氨基酸序列如图10所示。
7、毕赤酵母KAR2基因的克隆及载体构建
从NCBI数据库中获得毕赤酵母KAR2基因的DNA序列，设计如下引物进行基因扩增：(酶切位点以下划线标出)
KAR2Forward:CGGTTCGAAACGATGCTGTCGTTAAAACCATCT(SEQ ID NO:11)
KAR2Reverse:GCTGGCGGCCGCCTATGATCATGATGAGTTGTAG(SEQ ID NO:12)
以毕赤酵母GS115菌株基因组为模板，通过PCR反应获得KAR2基因，使用SnaB Ⅰ与Not Ⅰ双酶切消化，插入表达载体pPIC6(购自Invitrogen公司)，获得载体pPIC6-KAR2，其结构如图18所示。DNA序列测序正确，序列如图11所示，所对应的氨基酸序列如图12所示。
8、ERO1与rHSA共表达菌株的构建及筛选
以rHSA分泌表达菌株GS115-rHSA为原始菌株，上述构建好的pPICZα-ERO1载体经过Sac Ⅰ酶切线性化，电转化入GS115-rHSA菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA菌株染色体5’AOX位点。转化菌株采用含有2mg/mL博来霉素(Zeocin)的YPD固体培养基进行抗生素富集筛选，获得了ERO1与rHSA共表达的酵母菌株GS115-rHSA-ERO1。
9、HAC1与rHSA共表达菌株的构建及筛选
以rHSA分泌表达菌株GS115-rHSA为原始菌株，实施例4构建的pPIC6-HAC1载体经过Sac Ⅰ酶切线性化，电转化入GS115-rHSA菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA菌株染色体5’AOX位点。转化菌株采用含有1mg/mL杀稻瘟菌素(Blasticidin)的YPD固体培养基进行抗生素富集筛选，获得了HAC1与rHSA共表达的酵母菌株GS115-rHSA-HAC1。
10、PDI与rHSA共表达菌株的构建及筛选
以rHSA分泌表达菌株GS115-rHSA为原始菌株，上述构建好的pPICZα-PDI载体经过Sac Ⅰ酶切线性化，电转化入GS115-rHSA菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA菌株染色体5’AOX位点。转化菌株采用含有2mg/mL博来霉素(Zeocin)的YPD固体培养基进行抗生素富集筛选，获得了PDI与rHSA共表达的酵母菌株GS115-rHSA-PDI。
11、PPI与rHSA共表达菌株的构建及筛选
以rHSA分泌表达菌株GS115-rHSA为原始菌株，实施例6构建的pPIC6-PPI载体经过Pme Ⅰ酶切线性化，电转化入GS115-rHSA菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA菌株染色体5’AOX位点。转化菌株采用含有1mg/mL杀稻瘟菌素(Blasticidin)的YPD固体培养基进行抗生素富集筛选，获得了PPI与rHSA共表达的酵母菌株GS115-rHSA-PPI。
12、KAR2与rHSA共表达菌株的构建及筛选
以rHSA分泌表达菌株GS115-rHSA为原始菌株，实施例7构建的pPIC6-KAR2载体经过Pme Ⅰ酶切线性化，电转化入GS115-rHSA菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA菌株染色体5’AOX位点。转化菌株采用含有1mg/mL杀稻瘟菌素(Blasticidin)的YPD固体培养基进行抗生素富集筛选，获得了KAR2与rHSA共表达的酵母菌株GS115-rHSA-KAR2。
13、HAC1、PDI与rHSA共表达菌株的构建及筛选
以表达菌株GS115-rHSA-PDI为原始菌株，上述构建好的pPIC6-HAC1载体经过Sac Ⅰ酶切线性化，电转化入GS115-rHSA-PDI菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA-PDI菌株染色体5’AOX位点。转化菌株采用含有1mg/mL杀稻瘟菌素(Blasticidin)的YPD固体培养基进行抗生素富集筛选，获得了HAC1、PDI与rHSA共表达的酵母菌株GS115-rHSA-PDI-HAC1。
14、PPI、PDI与rHSA共表达菌株的构建及筛选
以实施例10筛选的表达菌株GS115-rHSA-PDI为原始菌株，实施例6构建的pPIC6-PPI载体经过Pme Ⅰ酶切线性化，电转化入GS115-rHSA-PDI菌株。感受态制备与电转化的方法均参照文献(James M.Cregg Pichia Protocols 2nd)。插入片段整合入GS115-rHSA-PDI菌株染色体5’AOX位点。转化菌株采用含有1mg/mL杀稻瘟菌素(Blasticidin)的YPD固体培养基进行抗生素富集筛选，获得了PPI、PDI与rHSA共表达的酵母菌株GS115-rHSA-PDI-PPI。
15、rHSA共表达菌株摇瓶诱导表达
分别挑取上述实施例中筛选的GS115-rHSA-ERO1、GS115-rHSA-HAC1、GS115-rHSA-PDI、GS115-rHSA-PPI、GS115-rHSA-KAR2、GS115-rHSA-PDI-HAC1及GS115-rHSA-PDI-PPI菌株单菌落，接种于2毫升MGY(1.34％酵母氮源碱；1.0％甘油；4.0×10-5生物素)培养基，30℃培养16小时，离心后收集菌体，转移至20毫升BMMY(1.0％酵母浸出物；2.0％蛋白胨；0.1M磷酸钾缓冲液，PH6.0；1.34％酵母氮源碱；0.5％无水甲醇)培养基培养，诱导表达72小时，其中每12小时补加50微升无水甲醇。诱导结束后取培养上清进行SDS-PAGE电泳检测(图19)。与对照菌株(GS115-rHSA)相比，7株共表达菌株rHSA的表达量都有提高，采用Quantity One软件分析，表达率如表1所示。
表1
菌株 表达量比率
GS115-rHSA 100％
GS115-rHSA-PDI 260％
GS115-rHSA-HAC1 210％
GS115-rHSA-KAR2 168％
GS115-rHSA-PPI 162％
GS115-rHSA-ERO1 150％
GS115-rHSA-PDI-HAC1 280％
GS115-rHSA-PDI-PPI 220％
16、rHSA共表达菌株的发酵
采用5升发酵罐对GS115-rHSA菌株以及实施例15筛选的GS115-rHSA-ERO1、GS115-rHSA-HAC1、GS115-rHSA-PDI、GS115-rHSA-PPI、GS115-rHSA-KAR2、GS115-rHSA-PDI-HAC1及GS115-rHSA-PDI-PPI菌株进行发酵，发酵条件参考Invitrogen公司公开的《Pichia Fermentation Process Guidelines》。诱导表达80小时后结束发酵，取培养上清分析rHSA的表达量。结果如表2显示，当固定发酵时间为80小时的情况下，共表达菌株rHSA的表达量明显提高，最高可达18.2g/L发酵上清，为rHSA大规模工业化生产奠定基础。
表2
菌株 最大表达量(g/L)
GS115-rHSA 5.98
GS115-rHSA-PDI 16.9
GS115-rHSA-HAC1 12.6
GS115-rHSA-KAR2 10.0
GS115-rHSA-PPI 9.7
GS115-rHSA-ERO1 8.9
GS115-rHSA-PDI-HAC1 18.2
GS115-rHSA-PDI-PPI 13.1

Claims (14)

  1. 高效表达重组人血清白蛋白的方法，其特征在于包括以下步骤：在酵母宿主细胞中，将(a)人血清白蛋白基因与(b)一种或多种rHSA表达促进因子基因进行共表达。
  2. 权利要求1的方法，其中所述酵母为毕赤酵母属(Pichia)。
  3. 权利要求1的方法，其中所述酵母为巴斯德毕赤酵母(Pichia pastoris)。
  4. 权利要求1-3之任一项的方法，其中所述rHSA表达促进因子选自由转录激活因子HAC1、结合蛋白KAR2、蛋白质二硫键异构酶(PDI)、内质网氧化还原酶(ERO1)和脯氨酸顺反异构酶(PPI)构成的组。
  5. 权利要求1-4之任一项的方法，其中1种、2种、3种或更多种rHSA表达促进因子基因被引入所述酵母宿主细胞。
  6. 权利要求1-5之任一项的方法，其中所述人血清白蛋白基因通过质粒转化入所述酵母宿主细胞。
  7. 权利要求1-6之任一项的方法，其中所述rHSA表达促进因子基因通过1个、2个或更多个质粒转化入所述酵母宿主细胞。
  8. 高效表达重组人血清白蛋白的工程菌，其中该工程菌为酵母，并且包含：(a)人血清白蛋白基因与(b)一种或多种rHSA表达促进因子基因。
  9. 权利要求9的工程菌，其中所述酵母为毕赤酵母属(Pichia)。
  10. 权利要求9的工程菌，其中所述酵母为巴斯德毕赤酵母(Pichia pastoris)。
  11. 权利要求8-10之任一项的工程菌，其中所述rHSA表达促进因子选自由转录激活因子HAC1、结合蛋白KAR2、蛋白质二硫键异构酶(PDI)、内质网氧化还原酶(ERO1)和脯氨酸顺反异构酶(PPI)构成的组。
  12. 权利要求8-10之任一项的工程菌，其中1种、2种、3种或更多种rHSA表达促进因子基因被引入所述工程菌。
  13. 权利要求8-12之任一项的工程菌，其中所述人血清白蛋白基因通过质粒转化入所述工程菌。
  14. 权利要求8-13之任一项的工程菌，其中所述rHSA表达促进因子基因通过1个、2个或更多个质粒转化入所述工程菌。
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