Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N -acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N -acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N -terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto- N -triose II (LNT II), lacto- N -tetraose (LNT), and sialyllacto- N -tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto- N -tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates. • Functional human 2,6-sialyltransferase hST6GalNAc V/VI were successfully expressed in Escherichia coli. • The recombinant hST6GalNAc V/VI displayed flexible substrate specificity and lower enzymatic efficiency. • Human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both hST6GalNAc V/VI. [ABSTRACT FROM AUTHOR]