β-内酰胺酶抑制短肽SIPIS04-01的表达、纯化与抑制作用测定

Expression, purification and inhibition assay of a β-lactamase inhibitory peptide SIPIS04-01

通过酵母双杂交系统从一个随机DNA片段文库中筛选到一个编码能与β-内酰胺酶结合的短肽SIPIS04-01的DNA序列,将它克隆到pGEX-4T-1的多克隆位点中,得到重组质粒pYG205。当用适量的IPTG诱导后,携带pYG205的E.coliDH5α能表达短肽SIPIS04-01-GST融合蛋白。利用谷胱甘肽Sepharose4B亲和层析介质分离纯化短肽SIPIS04-01-GST融合蛋白,经凝血酶切割并分离纯化后,体外试验表明短肽SIPIS04-01具有抑制β-内酰胺酶的作用。

A DNA fragment encoding a peptide SIPIS04-01 which can bind β-lactamase was obtained from a random DNA fragment bank by screening of yeast two-hybrid system, and further subcloned into pGEX-4T-1 to obtain a recombinant plasmid pYG205. SIPIS04-01-GST fusion protein was expressed from a recombinant E.coli DH5α containing plasmid pYG205 under the induction of a suitable amount of IPTG and purified by glutathione Sepharose 4B affinity chromatography. Finally, peptide SIPIS04-01 was isolated after cleavage from fusion protein with thrombin. It was showed that peptide SIPIS04-01 had a β-lactamase inhibiotry effect to some extent in vitro.