Heterologous expression of Trichoderma reesei exoglucanase (Cel6A) in Pichia pastoris under the control of GAP promoter
Trisanti AnindyawatiRaditya PutraYuliawati YuliawatiKartika Sari DewiAsrul Muhamad FuadYanni Sudiyani
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Cellulolytic microorganisms produce several cellulase enzymes which have different specificities and modes of action. At least three types of cellulase (endo, exo, and β-glucosidase) are involved in the degradation of cellulose. One of them, exo-β-1,4 glucanase or cellobiohydrolase, can release either glucose or cellobiose from ends of cellulose chains. In this study, we tried to express an exoglucanase (Cel6A) gene heterologically in Pichia pastoris. The Cel6A gene was derived from Trichoderma reesei cellobiohydrolase 2 (CBH2). It was synthetically prepared, and codon optimized for best expression in yeast P. pastoris. The gene was placed under the regulation of the GAP promoter. The recombinant plasmid, named pLIPI-TrCel6A, inserted with T. reesei Cel6A (TrCel6A) gene has been integrated into P. pastoris SMD1168H genome, and the recombinant enzyme has been successfully expressed by P. pastoris with a major product showing a molecular size of around 50 kDa.Keywords:
Heterologous
Heterologous expression
Objective To create protease-deficient mutants for improving productivity and secretion efficiency of heterologous protein expression.Methods PEP4 gene of Pichia pasioris was disrupted by transforming with pRS306/PEP4 LR vector.Effects of the resultant PEP4 strain on heterologous protein expression were then measured by practical expression compared with the wild strain.Results By disrupting the PEP4 gene in Pichia pastoris,the productivity of heterologous protein expression increased 9.5% on average.Conclusion Disruption of PEP4 gene in Pichia pastoris is an effective method for controlling proteolytic degradation of heterologous protein MIP.
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Pichia pastoris is a new eukaryotic expression system with great potentials, has unexampled advantages in expressing the heterologous proteins and has gotten more and more wide application. The following aspects were described in detail: the advantages of Pichia pastoris in the expression of heterologous genes, the mechanism of the heterologous gene integrations, the glycosylation of the expressed protein and the modification of the post-translated protein.
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Heterologous expression
Pichia
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Pichia
Heterologous expression
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Heterologous
Secretory protein
Folding (DSP implementation)
Heterologous expression
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Abstract Pichia pastoris was transformed with the Trichoderma reesei cbh1 gene, and the recombinant enzyme was purified and analyzed kinetically and by circular dichroism. The P. pastoris rCBH I was recognized by MoAb raised to T. reesei CBH I but was found in multiple molecular weight species on SDS−PAGE gels. Carbohydrate content determination and SDS−PAGE western analysis indicated that the recombinant protein was hyperglycosylated, although a species very similar in molecular weight to the T. reesei enzyme could be isolated chromatographically. The P. pastoris rCBH I also demonstrated activity toward soluble and insoluble substrates (i.e., pNPL and Sigmacell), although at a level significantly lower than the wild‐type enzyme. More seriously, the yeast‐expressed enzyme showed non‐wild‐type secondary structure by circular dichroism. We conclude that P. pastoris may not serve as an adequate host for the site‐directed mutagenesis of T. reesei CBH I.
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Several hundred heterologous proteins have been expressed in Pichia pastoris as the rapid progress of genic engineering. In this review, the advantages and the composings of Pichia pastoris expression system and the translated methods and the expressed characteristics of the expression system were summarized. The wide applications of producing heterologous protein in the system were expatiated. Effect factors of heterologous protein,s expression in the expression system and the strategies for optimizing the expression system were also summarized stressly.
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methylotrophic 酵母 Pichia pastoris 由于它为有效蛋白质表示的唯一的特征 / 能力是为许多 heterologous 蛋白质的生产的一个高度成功的系统，并且巨大的努力被作了由 P 增加 heterologous 蛋白质生产率。最近的年里的 pastoris。当新设计酵母种类被构造并且准备为工业蛋白质生产使用时，进程控制和优化技术应该被使用在下列方面改进发酵性能：(1 ) 在生长阶段期间在 fermentor 增加 recombinant 房间集中到高密度；(2 ) 有效地由在正式就职阶段期间提高 / 稳定蛋白质的 titers 或集中导致 heterologous 蛋白质；(3 ) 减少操作由减轻工作花费大量热交换和氧供应。这篇文章由 P 在 heterologous 蛋白质生产考察并且讨论关键、通常使用的技术。pastoris 与发酵媒介和基本操作条件的优化的焦点，为完成 P 的高密度耕作喂策略的最佳的甘油的开发。由调整特定的生长的 pastoris 和有效 heterologous 蛋白质正式就职方法评价，甲醇集中，温度，多碳底层的混合比率，等等。为由 P 的 recombinant 蛋白质生产的新陈代谢的分析。pastoris 也被介绍解释非最优的 heterologous 蛋白质生产的机制并且探索进一步最佳的表示方法。
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Pichia
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Pichia pastoris expression system is an ideal choice for the production of heterologous proteins,however,not all the heterologous proteins can be successfully and efficiently expressed in the host,there are distinct differences among protein yield levels,bioactivity and stability.The article summarized the main factors impacting the efficient expression of recombinant proteins in P.pastoris,and described the strategy for the efficient expression.
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Pichia
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It is very important to produce heterologous proteins both in theory and in practice,expecially for biopharmaceutical.Pichia pastoris has become one of the best eucaryotic expression systems in recent years.The factors influencing the high-level expression of heterologous protein in P.pastoris and the core strategies to hyperexpress heterologous proteins in P.pastoris were discussed in this paper.
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Biopharmaceutical
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Pichia pastoris is easy to genetically manipulate and can be grown to high cell densities,at the same time,P.pastoris is a eukaryote,and thereby has the potential for producing soluble,correctly folded recombinant protein with high yield,so the P.pastoris expression system is an ideal choice for the production of various heterologous proteins.However,at present not all the heterologous proteins can be successfully and efficiently expressed in P.pastoris,as a result,different proteins present different yield levels,bioactivity and stability.Here strategies for reducing proteolytic degradation and improving production of the expressed heterologous proteins are briefly summarized in terms of genetically factors and cultivation level.
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Heterologous expression
Eukaryote
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