Cold-shock Protein Expression System Facilitates the Solubility of Human ST6Gal I in Escherichia Coli

摘要

The protein structures of most mammalian sialyltransferases have yet to be elucidated. Practical and convenient protein expression systems for soluble and active sialyltransferases will facilitate elucidation of the protein structures and catalytic mechanisms of these enzymes. The present study was performed to establish an efficient expression system for human ST6Gal I (hST6Gal I). cDNA encoding a soluble form of hST6Gal I was introduced into the bacterial expression vector pCold I carrying the cold shock promoter that is inducible by low-temperature conditions. The resultant DNA en-codes the enzyme fused in frame with a maltose-binding protein (MBP) as a purification tag. This expression plasmid was introduced into the E. coli strain pGro7/BL21 harboring the molecular chaperones GroES and GroEL. Combined use of chaperone proteins and low-temperature cultivation during IPTG induction significantly improved the functional enzyme solubility in bacteria. The MBP-tagged hST6Gal I was efficiently purified by affinity chromatography using amylose-conjugated agarose.