Chondroitin sulfate C (CSC), a 6-O-sulfated chondroitin sulfate, is widely used in osteoarthritis treatment. However, the soluble expression and catalytic efficiency of Homo sapiens chondroitin 6-O-sulfotransferase (HsC6ST), a key enzyme for CSC biosynthesis, remain limited. Here, soluble expression of HsC6ST in Escherichia coli BL21(DE3) was achieved by fusion with the trigger factor (TF). To further improve its performance, a dynamic surface loop was deleted to generate TF-HsC6ST-Δloop. The mutant exhibited enhanced thermostability, with the half-life increasing from 5.91 to 9.44 h and the melting temperature (Tm) increasing from 88 °C to 97 °C. In addition, its catalytic efficiency (kcat/Km) toward PAPS and chondroitin increased by 1.4-fold and 1.9-fold, respectively. Molecular simulation suggested stronger ligand binding, with ΔGbind decreasing from -24.47 kcal/mol to -40.1 kcal/mol. Consequently, the CSC conversion rate increased from 41.2% to 78.4%.
Wang et al. (Mon,) studied this question.