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

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。

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

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.