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.