枯草芽孢杆菌噬菌体DNA甲基转移酶基因在大肠杆菌和枯草芽孢杆菌中的克隆与表达。

Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis.

作者信息

Günthert U, Reiners L, Lauster R

出版信息

Gene. 1986;41(2-3):261-70. doi: 10.1016/0378-1119(86)90106-x.

Abstract

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.

摘要

温和型枯草芽孢杆菌噬菌体SPR（野生型及各种突变体）、phi 3T、rho 11和SPβ的DNA甲基转移酶（Mtase）基因已在大肠杆菌和枯草芽孢杆菌宿主-质粒载体系统中克隆并表达。通过对无细胞提取物进行体外甲基化测定，已对这些克隆中的Mtase活性进行了定量。当从其天然启动子转录时，这四种噬菌体的Mtase基因合成的Mtase量有所不同。在枯草芽孢杆菌以及大肠杆菌中，SPR Mtase的产量总是比其他噬菌体Mtase少。SPR Mtase的表达水平取决于上游载体启动子序列的强度。通过与λ pL或tac启动子融合，在大肠杆菌中（诱导表达）实现了SPR野生型和突变酶的过量表达，通过噬菌体SP02启动子在枯草芽孢杆菌中（组成型表达）实现了过量表达。