分子伴侣协助下抗肿瘤抗生素美达霉素生物合成中的糖基转移酶Med-ORF8 的原核表达

Prokaryotic expression of a glycosyltransferase Med-ORF8 involved in an antitumor antibiotic medermycin biosynthesis aided by the molecular chaperone

摘要:

糖基转移酶在天然产物的糖基化修饰过程中起关键作用。目前链霉菌源抗肿瘤抗生素美达霉素(Medermycin)生物合成途径中的糖基转移酶Med-ORF8 的原核表达及酶学性质还未有研究。首先通过结构模拟确定Med-ORF8 的端部加上His-标签不会影响其三维结构的正确折叠, 然后利用2 种pET 原核表达载体来进行Med-ORF8 的原核表达, 发现以pET-28a (+)为载体进行表达时, 目的蛋白产量非常高, 但是以不可溶的包涵体形式为主。当分子伴侣基因(编码大肠杆菌触发因子)与Med-ORF8 的编码基因共表达时, 在优化诱导条件的情况下, 可以有效减少包涵体的形成,提高了Med-ORF8 的可溶性表达效率, 为Med-ORF8 的酶学分析打下基础。

Abstract:

Glycosyltransferases play central roles in the glycosylation of biosynthetic pathways of many antibiotics. The prokaryotic expression and enzymatic features of Med-ORF8, a glycosyltransferase involved in the biosynthesis of medermycin with strong antitumor activity, still remains obscure. Here, firstly, we performed the computer modeling of the 3-D structure of Med-ORF8 to prove that the presence of 6*His-tag at the terminals of Med-ORF8 had no effect on its 3-D structure. Subsequently, we established two prokaryotic expression systems with pET vectors to express Med-ORF8, and found that pET-28a (+) could gain a higher yield of the target protein than pET-23a (+), but mostly in an insoluble form. Finally, we introduced a molecular chaperone gene into the system with pET-28a (+) and found that the co-expression of the molecular chaperone with Med-ORF8 could efficiently decrease the formation of the inclusion body and increase the accumulation of soluble Med-ORF8.