大肠杆菌肽聚糖合成酶MraY在杆状病毒系统中的表达

Expression of peptidoglycan synthetase MraY of Escherichia coli in baculovirus expression system

磷酸-N-乙酰胞壁酸酯-胸腺喷丁转位酶(MraY)是参与大肠杆菌细胞壁合成的一种膜蛋白。由于该蛋白不能通过大肠杆菌表达系统进行过表达制备重组蛋白,本研究采用杆状病毒表达系统,通过PCR从大肠杆菌基因组中扩增出目的基因MraY,纯化后克隆于pFastBac 1质粒中构建重组转移质粒pFastBac-MraY。将其转化DH10感受态,构建重组杆粒Bacmid-MraY,并转染于Sf9昆虫细胞中进行重组杆状病毒制备和目的蛋白表达,采用western blot和间接免疫荧光鉴定结果表明,目的蛋白在昆中细胞Sf9中获得表达,分子量为41 ku并以可溶性形式表达。该蛋白的表达为MraY的高级结构的解析以及噬菌体溶菌机制的研究奠定了基础。

Phospho-Mur-N-Ac-pentapeptide translocase （MraY） is a membrane protein of E.coli which is considered to be associated with bacterial cytoderm synthesis. However, this protein is unable to be overexpressed in prokaryote system. To express MraY in eukaryote expression system, the MraY gene was amplified from E.coli by PCR and cloned into pFastBac 1 vector to construct the expression plasmids of pFastBac-MraY, which were transformed into DH10 to prepare recombinant Bacmid of Bacmid-MraY. Then the recombinant baculovirus was generated by transferring Bacmid-MraY into Sf9 insect cell, and the MmY expressed with a MW of 41 ku in soluble form detected by western blot and indirect immunofluorescence assay. The expression of this protein provided a basis for further study on the structure and MraY involved in the phage lysis mechanism.