Expression of highly active chondroitin 4-O-sulfotransferase-1 in Escherichia coli by a trigger factor fusion protein expression system

Abstract

Chondroitin 4-O-sulfotransferase-1 (C4ST-1) is a key enzyme for the biosynthesis of chondroitin sulfate A (CSA). Several past reports have evaluated the expression of C4ST-1 in bacteria. Since N-glycosylation is critical for the activity of C4ST-1, its activity was quite low. Here, we established a method for inducing a high expression of recombinant C4ST-1 in Escherichia coli (E. coli) by using a trigger factor (TF) fusion system. This approach enabled the expression of 18.9 ± 5.0 mg/L of TF-fused C4ST-1 (TF-C4ST-1). Although TF-C4ST-1 does not undergo N-glycosylation, the kcat/Km was approximately 60% of that of N-glycosylated C4ST-1. By using TF-C4ST-1 and chondroitin (CH) obtained from the culture of another recombinant E. coli, we succeeded in the 10-mg-scale preparation of CSA consisting almost entirely of the CSA disaccharide, GlcAβ1–3GalNAc(4S). These results demonstrated that the expression of C4ST-1 in bacteria is sufficient to achieve the long-term goal of manufacturing non-animal-derived CSA.