GL-7-氨基头孢烷酸酰化酶在大肠杆菌中的分泌表达

Secretory Expression of GL-7-ACA Acylase in E. coli.

作者信息

Chen Jian-Feng, Li Yong, Jiang Wei-Hong, Yang Yun-Liu, Zhao Guo-Ping, Wang En-Duo

机构信息

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031, China.

出版信息

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1998;30(4):393-396.

PMID:12168043

Abstract

The gene of beta-subunit of GL-7-ACA acylase [7beta-(4-carboxybutanamido) cephalosporanic acid acylase] was cloned into pTrc99B, an IPTG inducible pasmid, to form the recombinant called pTrc-CA1B. Another recombinant plasmid pTrcCA1S was obtained by cloning the gene encoding alpha-subunit of GL-7-ACA acylase, the signal peptide and the expression elements from Pseudomonas sp. into pTrcCA1B. Then, recombinant plasmid pKKCA1S was constructed by cloning the gene encoding the signal peptide, expression elements and GL-7-ACA acylase into the vector pKK235. pTrcCA1S and pKKCA1S were allowed to transform TG1. These two plamids were able to transfer the expression product into the periplasmic space of the host bacteria. As a result, in the whole cell of TG1/pTrcCAIS, the specific activity of GL-7-ACA acylase was 23.9 u/g cell, 8 fold higher than that of TG1/pMR24. And in the whole cell of TG1/pKKCA1S, the specific activity of acylase was 18.3 u/g cell, 6 fold higher than that of TG1/pMR24.

摘要

将GL-7-ACA酰化酶[7β-(4-羧基丁酰胺基)头孢菌素酸酰化酶]β亚基的基因克隆到pTrc99B（一种IPTG诱导型质粒）中，构建重组体pTrc-CA1B。通过将GL-7-ACA酰化酶α亚基、信号肽及来自假单胞菌属的表达元件的编码基因克隆到pTrcCA1B中，获得另一个重组质粒pTrcCA1S。然后，将信号肽、表达元件及GL-7-ACA酰化酶的编码基因克隆到载体pKK235中，构建重组质粒pKKCA1S。用pTrcCA1S和pKKCA1S转化TG1。这两种质粒能够将表达产物转运到宿主菌的周质空间。结果，在TG1/pTrcCA1S的全细胞中，GL-7-ACA酰化酶的比活性为23.9 u/g细胞，比TG1/pMR24高8倍。在TG1/pKKCA1S的全细胞中，酰化酶的比活性为18.3 u/g细胞，比TG1/pMR24高6倍。