Abstract:Chondroitin-4-O-sulfotransferase-1（C4ST-1, EC 2.8.2.5） catalyzes the sulfation of 4-OH of GalNAc to generate chondroitin sulfate A（CSA）. C4ST-1 contains three pairs of disulfide bonds and is hard to correctly fold into active form. As a result, C4ST-1 mainly exists as inclusion bodies in E. coli. To increase the soluble expression, the effects of co-expression of thiol oxidase (Erv1p) and C4ST-1 or C4ST-1 and disulfide isomerase (DsbC) were investigated. The results showed that co-expression of DsbC significantly increased the expression level of intracellular soluble protein while no significant effect was observed for co-expression of Erv1p. Co-expression of C4ST-1 and Erv1p, or C4ST-1 and DsbC increased the enzyme activities to （12.32±0.76） U/L and （21.99±0.42） U/L, which were 1.30 and 2.33 times of that of the original strain, respectively. Then, C4ST-1, Erv1p and DsbC were co-expressed and the enzyme activities in shake flask and 3 L fermenter were improved to （29.12±0.66） U/L and 49.97 U/L, respectively. The present study provides a solid foundation for further applications.
