大肠杆菌可溶性细胞色素b562编码基因的克隆与表达。

Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli.

作者信息

Nikkila H, Gennis R B, Sligar S G

机构信息

Department of Biochemistry, University of Illinois, Urbana 61801.

出版信息

Eur J Biochem. 1991 Dec 5;202(2):309-13. doi: 10.1111/j.1432-1033.1991.tb16377.x.

DOI:10.1111/j.1432-1033.1991.tb16377.x

PMID:1761034

Abstract

The gene for the soluble cytochrome b562 from Escherichia coli B has been cloned on a SalI fragment. The analysis of the gene reveals the presence of a leader sequence in front of the sequence encoding the mature protein. Expression of cytochrome b562 using the lac-promoter produced the protein to a level of 3-5% of total protein. This over-production enables employment of a simple, high-yield purification protocol to obtain homogeneous cytochrome b562. Spectroscopic and N-terminal sequence analyses of the purified protein demonstrate that it is identical to the chromosomally expressed cytochrome b562 purified and characterized from E. coli B [Itagaki, E. & Hager, L.P. (1966) J. Biol. Chem. 241, 3687-3695]. It is demonstrated that the genomic sequence codes for a classic N-terminal signal sequence and that mature cytochrome b562 is translocated to the periplasmic space.

摘要

来自大肠杆菌B的可溶性细胞色素b562基因已克隆在一个SalI片段上。对该基因的分析表明，在编码成熟蛋白的序列之前存在一个前导序列。使用lac启动子表达细胞色素b562，该蛋白产量达到总蛋白的3% - 5%。这种过量表达使得能够采用简单、高产的纯化方案来获得均一的细胞色素b562。对纯化蛋白的光谱分析和N端序列分析表明，它与从大肠杆菌B中纯化和鉴定的染色体表达的细胞色素b562相同[板垣英，哈格，L.P.（1966年）《生物化学杂志》241，3687 - 3695]。结果表明，基因组序列编码一个典型的N端信号序列，成熟的细胞色素b562被转运到周质空间。