Ferreira L C, Schwarz U, Keck W, Charlier P, Dideberg O, Ghuysen J M
Abteilung Biochemie, Institut für Entwicklungsbiologie, Tübingen, Federal Republic of Germany.
Eur J Biochem. 1988 Jan 15;171(1-2):11-6. doi: 10.1111/j.1432-1033.1988.tb13751.x.
Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.
通过改变dacA基因的羧基编码末端，获得了羧基末端截短的大肠杆菌青霉素结合蛋白5（PBP5）衍生物。将修饰后的dacA基因克隆到失控复制控制质粒中后，选择一个过量产生所需蛋白质并将其分泌到周质中的克隆作为分离水溶性PBP5（即PBP5S）的来源。在PBP5S中，野生型蛋白的羧基末端21个氨基酸区域被一个短的9个氨基酸片段取代。在不使用去污剂或离液剂的情况下，通过青霉素亲和层析法纯化了毫克量的PBP5S。PBP5S很稳定，在不添加 Triton X-100的情况下具有DD-羧肽酶活性。与PBP5一样，与[14C]苄青霉素反应时，它会转化为一种寿命较短的酰基酶复合物。PBP5和PBP5S都结晶了。与PBP5不同，PBP5S产生了适合X射线分析的具有酶活性的、形状良好的棱柱形晶体。
