一种通过过表达毕赤酵母翻译相关因子提高重组蛋白胞内表达的策略  

A Strategy to Increase the Intracellular Overexpression of Recombinant Proteins by Expressing Pichia pastoris Translation-related Factors

作　　者：林雯炀 廖锡豪 陈南柱 黄大富 陈亮[1] 钟炳旭 梁书利[1] 林影[1] LIN Wen-yang;LIAO Xi-hao;CHEN Nan-zhu;HUANG Da-fu;CHEN Liang;ZHONG Bing-xu;LIANG Shu-li;LIN Ying(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,China)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006

出　　处：《现代食品科技》2021年第7期66-73,共8页Modern Food Science and Technology

基　　金：国家自然科学基金资助项目(31470159)。

摘　　要：本研究为筛选对毕赤酵母重组蛋白表达有促进作用的翻译相关因子,扩增了毕赤酵母中包括核糖体蛋白翻译起始因子、翻译延伸因子、氨酰-tRNA合成酶、核糖体生物合成因子、核糖体分子伴侣五类翻译相关因子(共28个)的基因片段,并将上述基因片段与质粒pPICZA进行同源重组,进一步获得重组载体后分别转化至以增强型绿色荧光蛋白eGFP为报告蛋白的毕赤酵母中。之后通过测量计算120 h内28个过表达翻译相关因子菌株的荧光蛋白表达量,筛选对毕赤酵母重组蛋白表达有潜在促进作用的因子,进一步以红色荧光蛋白mRFP为报告蛋白进行验证。结果表明:本研究共筛选到了6个对毕赤酵母重组蛋白胞内表达有促进作用的翻译相关因子eIF4A、eEF1A、eEF3、Ded81、Bcy1、Ssb。其中eIF4A、eEF1A、eEF3、Ded81、Bcy1、Ssb的eGFP单位菌体表达量分别增加了18.40%、18.80%、29.50%、28.45%、21.60%,mRFP单位菌体表达量分别增加了20.00%、8.00%、19.00%、5.40%、15.40%。Bcy1使得eGFP表达菌株生物量增加20.00%,mRFP表达菌株生物量增加30.90%。为从翻译层面提高毕赤酵母重组蛋白表达提供了思路。This study was to screen the translation-related factors that promote the expression of recombinant proteins in Pichia pastoris,and amplify gene fragments of five types of translation-related factors(a total of 28)in Pichia pastoris(including ribosomal proteins,translation initiation factors,elongation factors,aa-tRNA synthetases,ribosome biogenesis factors,and ribosome-associated chaperones).These gene fragments were cloned into the pPICZA vector through homologous recombination.The recombinant vectors were further obtained and transformed into the Pichia pastoris GS115 strain with enhanced green fluorescent protein(eGFP)as the reporter protein.Then,the expression levels of 28 strains with overexpressed translation-related factors within 120 h were measured and calculated,to identify the key factors that could potentially promote the expression of the recombinant proteins in Pichia pastoris.Finally,the red fluorescent protein,monomeric red fluorescent protein(mRFP),was used as the reporter protein for verification.The results revealed that 6 translation-related factors(eIF4A,eEF1A,eEF3,Ded81,Bcy1,Ssb)that could improve the intracellular expression of recombinant proteins in Pichia pastoris,were screened in this study.Among them,the eGFP unit cell expression of eIF4A,eEF1A,eEF3,Ded81,Bcy1,Ssb increased by 18.40%,18.80%,29.50%,28.45%and 21.60%,respectively,and the mRFP unit cell expression increased by 20%,8.00,19.00%,5.40%and 15.40%,respectively.Bcy1 increased the biomass of eGFP-expressing strains by 20.00%and the biomass of mRFP-expressing strains by 30.90%.These results provide ideas for improving the expression of recombinant proteins in Pichia pastoris through the translation.

关 键 词：毕赤酵母 翻译相关因子 重组蛋白 胞内表达 

分 类 号：TS201.2[轻工技术与工程—食品科学]