高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (10): 2334-2339.doi: 10.7503/cjcu20130337

• 有机化学 • 上一篇    下一篇

信号肽对OPMA在枯草杆菌中的分泌表达及其催化活性的影响

李雁飞1, 张金祥1, 谷艳芹1, 郝倩1, 曹昊1, 张应玖1,2   

  1. 1. 吉林大学分子酶学工程教育部重点实验室, 长春 130012;
    2. 吉林大学生命科学学院, 长春 130012
  • 收稿日期:2013-04-15 出版日期:2013-10-10 发布日期:2013-10-10
  • 作者简介:张应玖,女,博士,教授,博士生导师,主要从事生物化学的研究.E-mail:yingjiu@jlu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号: 31170759, 31000053)资助.

Effects of Signal Peptide on the Secretory Expression of Multifunctional Amylase OPMA and Its Catalytic Activity

LI Yan-Fei1, ZHANG Jin-Xiang1, GU Yan-Qin1, HAO Qian1, CAO Hao1, ZHANG Ying-Jiu1,2   

  1. 1. Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, China;
    2. College of Life Science, Jilin University, Changchun 130012, China
  • Received:2013-04-15 Online:2013-10-10 Published:2013-10-10

摘要:

将编码枯草杆菌BS168的amyE信号肽(S)的基因与编码多功能淀粉酶OPMA-N和OPMA-G的基因重组, 分别构建了分泌表达多功能淀粉酶OPMA-N和OPMA-G的重组载体pMA5-OPMA-SN和pMA5-OPMA-SG, 并分别在枯草杆菌BS168中实现了OPMA-SN和OPMA-SG的有效分泌表达.SDS-PAGE分析表明, 该信号肽的引入提高了异源蛋白的分泌量, OPMA-SN和OPMA-SG的分泌水平(53%和67%)比OPMA-N和OPMA-G(45%和58%)明显提高, 但此信号肽在分泌表达OPMA-N或OPMA-G后不能被有效切除.活力分析表明, OPMA-SN(5.17 U/mg)和OPMA-N(4.57 U/mg)的活力水平与分泌水平一致, 但OPMA-SG(4.50 U/mg)和OPMA-G(4.65 U/mg)的活力水平却与分泌水平呈反相关性.通过分析OPMA-SN和OPMA-SG分子的空间构象发现, 信号肽的存在不影响OPMA-N的活性部位构象, 但影响OPMA-G的活性部位构象, 从而导致OPOMA-G的催化活性下降.

关键词: 多功能淀粉酶, 表达, 信号肽, 结构, 功能

Abstract:

The recombinant expression vectors pMA5-OPMA-SN and pMA5-OPMA-SG were constructed by fusing the gene encoding the signal peptide(S) of amyE from Bacillus subtilis 168 with the gene encoding the multifunctional amylases OPMA-N or OPMA-G, and the high-level secretory expression of OPMA-N and OPMA-G were achieved by Bacillus subtilis 168. SDS-PAGE analysis showed that the introduction of the signal peptide was indeed possible to improve the secretion of heterologous protein, and the secretory yields of OPMA-SN or OPMA-SG(53% or 67%) were obviously higher than those of OPMA-N or OPMA-G(45% or 58%), but the signal peptide could not be effectively removed after secretion. The activity analysis demonstrated that the levels of OPMA-SN and OPMA-N activities(5.17 and 4.57 U/mg) were consistent with their secretory yields, but it was not the case for OPMA-SG(4.50 U/mg) and OPMA-G(4.65 U/mg), there was a negative correlation between the activity levels and secretory yields of for OPMA-SG and OPMA-G. The spatial conformation of OPMA-SN and OPMA-SG molecules proved that the presence of the signal peptide at the N terminus of OPMA-N or OPMA-G could not affect the conformation of the catalytic centre in OPMA-N, but affected that in OPMA-SG and resulted in the decrease of OPMA-SG's catalytic activity. These findings could provide important guidance on signal peptide application for secretory expression of heterologous proteins in Bacillus subtilis, and for the structure and function of the target protein.

Key words: Multifunctional amylase, Expression, Signal peptide, Structure, Function

中图分类号: