分子伴侣对白喉毒素无毒突变体CRM197重组蛋白在大肠杆菌中可溶性表达的促进作用

为了获得有活性的白喉毒素突变体蛋白 (Cross-reacting material 197,CRM197),本研究利用分子伴侣pG-KJE8与重组质粒pET28a-CRM197在大肠杆菌原核表达系统中进行共表达,来促进目的蛋白的正确折叠,进而提高CRM197蛋白的可溶性表达。将质粒转化至大肠杆菌后并诱导其表达目的蛋白,再通过SDS-PAGE胶染色、Western blotting等技术对所得蛋白进行检测分析。结果发现：利用体外重组技术成功得到了pET28a-CRM197重组蛋白原核表达质粒,且CRM197重组蛋白在原核表达系统中主要以包涵体形式表达;通过探索和优化,确定了诱导蛋白的最佳浓度和温度,当加入终浓度为1.0 mmol/L IPTG、0.5 mg/mL L-阿拉伯糖、5.0 ng/mL四环素,在20 ℃条件下诱导16 h时,目的蛋白的可溶性表达得到显著提高;可溶性表达的CRM197重组蛋白可以与CRM197一抗发生特异性结合,免疫反应性良好。因此,研究发现分子伴侣pG-KJE8可以促进CRM197重组蛋白在大肠杆菌中以可溶性形式表达,且能很好地与CRM197一抗发生特异性结合,证实CRM197重组蛋白具有良好的免疫反应性,为CRM197蛋白的工业化生产及应用奠定了一定的基础。     

肝癌相关抗原SMP30重组基因的构建、表达及分子伴侣共表达系统的探索

旨在通过应用基因工程的方法构建、表达和纯化肝癌相关抗原SMP30,研究共表达分子伴侣提高基因工程蛋白表达的可溶性及效率。PCR扩增SMP30 cDNA序列,用基因工程技术构建重组表达质粒,转化E.coliBL21(DE3)pLysS宿主菌。表达蛋白经Ni-NTA亲和柱纯化获得HIS-SMP30融合蛋白;分别将4种表达不同分子伴侣的质粒(pG-KJE8、pGro7、pKJE7、pTf16)转入E.coliBL21(DE3)中;然后再将重组质粒转入含有分子伴侣质粒的细胞中,进行分子伴侣与重组质粒的共表达,SDS-PAGE检测目的蛋白的表达量与可溶性分析。经优化表达条件后,目的蛋白以包涵体形式表达,目的蛋白占总蛋白的60%以上;纯化后纯度高达95%以上;诱导共表达后,目的蛋白在上清含量极少,不到总表达目的蛋白的10%。成功构建出高效表达的SMP30重组质粒;加入到诱导表达体系中的4种分子伴侣质粒不能有效的促进可溶性蛋白的表达,pTf16共表达系统能增加目的蛋白表达量。     

共表达伴侣蛋白对重组毕赤酵母产米根霉(Rhizopus oryzae)脂肪酶发酵过程的影响

[目的]通过共表达伴侣蛋白Erolp和PDI获得米根霉(Rhizopus oryzae)脂肪酶在毕赤酵母中的高效表达.[方法]利用毕赤酵母基因重组菌高密度发酵的方法,在7L发酵罐水平上分析共表达伴侣蛋白菌株(BH128)与非共表达伴侣蛋白菌株(H238)对脂肪酶表达的差异.[结果]在相同条件下,BH128发酵产酶能力高于H238,最高酶活可达到2 338.7U/mL,最大比生长速率达到0.02 h-1,最大产物比形成速率达到944.5 U/(gDCW·h),最大底物比消耗速率也能达到0.15 gmethanol/(gDCW·h),分别是H238的1.7、0.5、4.1和1.3倍,且发酵周期缩短了20h.[结论]毕赤酵母基因重组菌BH128通过共表达伴侣蛋白Ero1p和PDI,提高了米根霉脂肪酶的产量,而且缩短了发酵周期,为工业化生产奠定了基础.     

Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli

Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS-PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS-PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.     

The chaperone activity of trigger factor is distinct from its isomerase activity during co-expression with adenylate kinase in Escherichia coli

To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co-expression. When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies. Co-expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Co-expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell. Co-expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl-prolyl cis/trans isomerase activity was 1% of wild-type, gave the same results as wild-type TF. This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.     

表达大肠杆菌分子伴侣GroEL、GroES和GrpE载体的构建及其应用

大肠杆菌（Escherichia coli）共表达系统常要求质粒具有不同抗生素抗性以及不同的复制子。利用粘性末端PCR技术,以含有大肠杆菌分子伴侣基因GroEL、GroES和唧E的pR—GESP质粒为模板,设计两对引物,通过两次独立的PCR反应扩增3个基因的多顺反子,将形成粘性末端的PCR产物插入NcoI和Xho1酶切的pACY．CDuet-1质粒,构建的pA—GESP质粒具有p15A复制子及氯霉素抗性,和具有ColE1复制子及卡那霉素抗性表达载体pET28b相容。SDS—PAGE显示含有pA—GESP质粒的大肠杆菌细胞中3个分子伴侣蛋白的表达水平和含有pR—GESP质粒的大肠杆菌细胞没有明显差异,它们对玉米丝氨酸消旋酶的可溶性表达有部分促进作用,但对N端含有组氨酸标签的玉米铁氧还蛋白还原酶的表达没有作用,在三个含有不同抗生素基因的质粒中共表达分子伴侣、5-氨基乙酰丙酸合酶和尿卟啉原III甲基化酶,两个酶连续催化的荧光产物在细胞内积累量为562．13±3．17／OD600,而没有分子伴侣的积累量为457．66±4．98／OD600,表明分子伴侣改善部分蛋白在大肠杆菌的可溶性表达和催化功能。     

In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E.coli

ABSTRACT: BACKGROUND: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E.coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. RESULTS: In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E.coli BL21/pET28a-lipAB and two dual expression plasmid systems E.coli BL21/pETDuet-lipA-lipB and E.coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E.coli BL21/pET28a-lipAB and E.coli BL21/pETDuet-lipA-lipB were 1300U/L and 3200U/L, respectively, while the activity of the lipase expressed by E.coli BL21/pACYCDuet-lipA-lipB was up to 8500U/L. The lipase LipA had an optimal temperature of 30[degree sign]C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. CONCLUSIONS: The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.     

共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

为了提高甘露聚糖酶ManA在毕赤酵母中分泌表达的酶活,选择毕赤酵母内质网未折叠蛋白反应(Unfolded protein response,UPR)激活调控因子HAC1与5种毕赤酵母蛋白折叠相关的分子伴侣ERO1、PDI、PDI1、CPR5、BiP,通过构建pPICZA-HAC1等6种胞内表达重组质粒,分别电转化至分泌表达ManA的毕赤酵母重组菌中胞内共表达,并分析其重组菌摇瓶发酵时ManA表达的影响。结果发现在摇瓶发酵水平,胞内共表达HAC1、ERO1、PDI的重组菌发酵上清液中的ManA酶活力分别提高了26%、15%、20%,其重组菌发酵上清液的酶活力分别达到1 014 U/mL、925 U/mL、965 U/mL。通过对各重组菌上清液酶活力、胞内滞留酶活力、上清液蛋白浓度数据进行分析,进一步选择将HAC1、ERO1、PDI进行两基因或三基因组合,并分别在分泌表达ManA的重组菌胞内共表达,但各共表达重组菌发酵上清液的酶活力都没有进一步的提升。单独共表达HAC1或者分子伴侣ERO1、PDI可以辅助ManA的正确折叠,提高其蛋白表达。     

分子伴侣共表达对嗜热环糊精葡萄糖基转移酶异源可溶性表达的影响

【目的】通过优化表达条件,提高嗜热环糊精葡萄糖基转移酶(CGTase)的可溶性表达和胞外酶活性。【方法】构建含cgt基因的重组表达质粒p ET-28a(+)-omp A-cgt,筛选最适诱导温度,并构建5种分子伴侣共表达系统(p KJE8、p KJE7、p Gro7、p Tf16和p G-Tf2,5种分子伴侣质粒分别与重组表达质粒p ET-28a(+)-omp A-cgt共表达),筛选最适分子伴侣质粒,优化共表达条件。【结果】通过SDS-PAGE分析和测定胞外酶活,CGTase基因在大肠杆菌中实现表达,且具有一定量的重组CGTase分泌至胞外;25°C诱导时CGTase的可溶性表达和在胞外上清中的酶活都最高;分子伴侣质粒p KJE8使酶的胞外活性提高了48.6%,效果最为显著;当L-阿拉伯糖浓度为0.5 g/L时,分子伴侣质粒p KJE8使酶的胞外活性提高了68.5%。【结论】通过优化表达条件及使用分子伴侣共表达系统提高了环糊精葡萄糖基转移酶的可溶性表达和胞外酶活,为该酶进一步相关研究奠定了基础。     

Co-expression of the small heat shock protein, Lo18, with β-glucosidase in Escherichia coli improves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES

We developed a new system to improve the overproduction of soluble proteins in E. coli based on a plasmid encoding the small heat-shock protein, Lo18, derived from the lactic acid bacterium Oenococcus oeni. The efficiency of this system was compared with that of another system based on production of the E. coli universal chaperone GroEL/ES. A compatible plasmid encoding β-glucosidase was constructed for the overproduction and aggregation of this enzyme. Co-expression with Lo18 resulted in an increase in soluble β-glucosidase levels similar to that obtained in the GroEL/ES co-expression system. Lo18 was found preferentially in the insoluble fraction, associated with aggregated enzyme. By contrast, GroEL/ES was more abundant in the soluble fraction.     

Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES

ABSTRACT: BACKGROUND: D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT. RESULTS: Intracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold. CONCLUSIONS: This is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.     

Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA

Overproduction of genetically engineered antibodies, such as single-chain antibodies (scAbs) in Escherichia coli often results in insoluble and inactive products known as inclusion bodies. We now report that fusion or co-expression of FkpA, the E. coli periplasmic peptidyl-prolyl-isomerase with chaperone activity, substantially improves soluble and functional expression of scAbs. Anti-human bladder carcinoma scAb (PG) and anti-human CD3 x anti-human ovarian carcinoma-bispecific scAb (BH1) were fused with FkpA on the pTMF-based plasmid and expressed in E. coli. More than half of the amount of each expressed fusion protein FkpA-PG or FkpA-BH1 was soluble. In addition, the fusion protein cellulose-binding domain from Cellulomonas fimi (CBD)-PG and anti-human CD3 x anti-human CD28 x anti-human ovarian carcinoma-trispecific scAb (TRI) fused to the pelB (a signal peptide from pectate lysase B of a Bacillus sp.) signal sequence were co-expressed with FkpA under the control of the T7 promoter. A substantial portion of the co-expressed CBD-PG or TRI was soluble. Furthermore, PG, BH1, and TRI were biologically active as judged by ELISA and in vitro cytotoxicity assay. These results suggest that overexpression of FkpA should be useful in expressing heterologous proteins in E. coli.     

hTNFR1在大肠杆菌中可溶性表达及纯化

将人源肿瘤坏死因子Ⅰ型受体（hTNFR1）基因克隆到pET-22b表达载体,成功构建了重组表达质粒pETH1,电转到Escherichia coli BL21（DE3）表达菌株中进行摇瓶发酵。实现了hTNFR1在大肠杆菌表达系统中的重组表达。但目的蛋白全部以包涵体的形式存在于沉淀中。为了提高hTNFR1在大肠杆菌中的可溶性表达,融合标签和分子伴侣两种策略被实施用于辅助hTNFR1的可溶性表达。结果表明,在hTNFR1的N端融合NusA标签后,hTNFR1的可溶性有一定提高;在NusA-hTNFR1基础上,过表达了7种分子伴侣,筛选出tig分子伴侣对hTNFR1蛋白可溶性表达有明显的促进作用,可溶性表达量约占总量的90％;对优化后的hTNFR1表达系统的可溶性蛋白进行Ni-NTA亲和层析纯化后,TEV蛋白酶酶切去除N端的NusA标签,结合Western blot分析鉴定,获得了大量高纯度的hTNFR1蛋白。研究结果为进一步研究hTNFR1的生理学活性及其在疾病治疗方面的应用奠定了良好基础。     

Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.     

Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli

An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL. On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library. A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned. Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E. coli), recombinant ALDH2 and ALDH2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E. coli chaperonin proteins. Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively. Co-expression of ALDH2 with ALDH2(2) in the presence of E. coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer. Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation. Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system. The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly. Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.     

Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli

Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.     

芽孢杆菌β-折叠桶植酸酶的原核可溶性表达优化及包涵体复性研究

目的:来源于芽孢杆菌的β-折叠桶植酸酶基因PhyH,截去N端120个碱基编码的40个氨基酸后,成功构建了原核表达体系,通过两种方法分别得到有活性的目的蛋白PhyHT,并通过进一步纯化提高目的蛋白的纯度.方法:通过分子伴侣共表达系统提高目的蛋白的可溶性表达,并通过包涵体复性研究,从包涵体中制备出有活性的目的蛋白.结果:(1)目的蛋白PhyHT主要以包涵体形式存在于沉淀中;(2)通过优化表达条件,降低温度和诱导剂浓度均不能明显改善包涵体问题,通过构建分子伴侣共表达系统(即pG-KJE8、pGro7、pKJE7和pTfl6 4种分子伴侣质粒分别与重组表达质粒pET28b-PhyHT共表达),筛选能提高目的蛋白可溶性表达的分子伴侣质粒;(3)包涵体经过复性和进一步的纯化,得到了高纯度的有生物活性的目的蛋白.     

Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli

Eight combinations of molecular chaperones (e.g., DnaK/DnaJ/GrpE/ClpB) are co-expressed with the target recombinant protein to compare their effectiveness in improving its soluble yield. This system allows the most complete and rational approach proposed so far to use the chaperone activity for optimizing the host cell folding machinery. Furthermore, a two-step protocol is presented, in which protein synthesis and protein refolding are uncoupled. Molecular chaperones and target protein accumulate in the first growth phase and target protein aggregates are then disaggregated in vivo after the block of protein synthesis. The optimal chaperone combination to maximize the soluble yield of a specific protein remains unpredictable. Therefore, a small-scale purification selection step is useful for screening among expression combinations before scaling-up production. Applying such a strategy, we could increase the solubility of 70% of the tested constructs with yields of up to 42-fold more protein than in controls. The procedure takes 2 d.     

费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域,其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达,探究其合适的表达及发酵条件,提高产量,降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后,应用pPIC9k质粒整合到毕赤酵母GS115基因组上,构建高产脂肪酶Lip605的毕赤酵母工程菌;并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法,综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为:甲醇3.103%(体积比),生物素0.4 mg/L,酵母粉11.5 g/L,酵母基础氮源培养基(yeast nitrogen base,YNB) 13.4 g/L,初始pH 6.4,装液量50 mL/250 mL,转速220 r/min,温度24°C,培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL,较优化前提高了5.8倍;进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达,其中共表达伴侣蛋白Rpl10(pPICZA-RPL10)效果最佳,可使Lip605表达量进一步提高46.8%;在此基础上,经过10 L发酵罐分批补料的高密度发酵,工程菌株发酵142 h,胞外脂肪酶酶活最高达到680 U/mL,蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量,为其进一步工业化生产奠定了良好的基础。     

DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli

Overexpression of CorA, the major magnesium transporter from bacterial inner membrane, in Escherichia coli resulted in the synthesis of 60mg of protein per liter of culture, most of which however was in the form of inclusion bodies. The levels of inclusion body formation were reduced by lowering the cell culture temperature. To dissect CorA inclusion body formation and the folding process involved, we co-expressed the protein with various chaperones and other folding modulators. Expression of DnaK/DnaJ (Hsp70) prevented inclusion bodies from forming and resulted in the integration of more CorA into the membrane. GroEL/GroES (Hsp60/Hsp10) were less effective at reducing CorA inclusion body formation. Co-expression with either Ffh/4.5S-RNA, the signal recognition particle, or SecA, the ATPase that drives protein insertion into the membrane, had little effect on CorA folding. These results indicate: (1) that CorA inclusion bodies form immediately after synthesis at 37 degrees C, (2) that CorA solubility in the cytosol can be increased by co-expressing a chaperone system, (3) membrane targeting is probably not a rate-limiting factor, and (4) that membrane insertion becomes a limitation only when large amounts of soluble CorA are present in the cytosol. These co-expression systems can be used for producing other membrane proteins in large quantities.