经密码子优化的耐热纤维素酶在大肠杆菌中的表达.pdfVIP

江苏农业学报(JiangsuJ.of Agr.Sci. ),2014,30(3):497 ~502 http:/ / 497 彭静静. 经密码子优化的耐热纤维素酶在大肠杆菌中的表达[J].江苏农业学报,2014,30(3):497-502. doi:10.3969/ j.issn.1000-4440.2014.03.007 经密码子优化的耐热纤维素酶在大肠杆菌中的表达 彭静静 (泰山学院生物与酿酒工程学院,山东 泰安 271021) 摘要: 为了提高质粒pHsh-celD在大肠杆菌中的表达量,运用 mRNA 二级结构预测软件对pHsh-celD 翻译起 始区的二级结构进行优化,得到了具有最佳 mRNA 二级结构及自由能的质粒pHsh-celD I。 为了进一步提高纤维素 酶的表达量,在不改变目的基因翻译的氨基酸序列的前提下,celD 的N-端氨基酸尽可能地使用大肠杆菌同义密码 子,定点突变后得到质粒pHsh-celD II。 优化后质粒pHsh-celD II 表达的纤维素酶活[(6.4±0.4) U/ ml]是未优化 [(4.1±0.3) U/ ml]的1.6倍。 SDS结果显示,该重组酶的分子量为66000,与理论值相符。 基于热激载体 pHsh 的重组表达系统具有诱导表达简便、诱导方式廉价的优点,且重组酶热稳定性好,这对该酶的大规模发酵应 用具有重要意义。 关键词: 纤维素酶; mRNA 二级结构; 优化; 热激载体 中图分类号: S634.3 文献标识码: A 文章编号: 1000-4440(2014)03-0497-06 Expression of thermo-stable cellulase gene subjected to optimization of mRNA secondary structure and codon usage inEscherichia coli PENGJing-jing (College of Biology and Enology,Taishan University,Taian271021,China) Abstract: In order to improve the expression level of celD gene, through structural optimization of plasmid pHsh-celD,the plasmid pHsh-celD I,which possessed the most appropriate structure and free energy of mRNA,was ob- tained. Higher expression level was achievedby replacingtherare codonsfor N-terminal amino acidsof thetarget protein, and the ultimateplasmid,pHsh-celDII wasresulted. Theexpressionlevel ofcelD wasincreasedfrom (4.1±0.3)U/ mlto (6.4±0.4) U/ ml in LB medium. SDSanalysis showed that the molecular mass of the expressed recombinant celD was about66000,exactly the sizepredicted.The expressionvector system of theheat shockplasmidpHsh owned advanta- ges such as high expression level and cheap induction. Moreover,the superior stability of therecombinant enzymelaidthe basefor the application of large-scalefermentation. Key words: cellulase; mRNA secondary structure; optimi