Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

Abstract

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13 degrees C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac alpha 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates.

Oral administration of a fusion protein containing eight GLP-1 analogues produced in Escherichia coli BL21(DE3) in streptozotocin-induced diabetic rats

“…Previous studies with induced protein expression in E. coli indicated that decreasing the culture temperature and using a longer induction time markedly increased target protein expression (Weickert et al 1997;Kagawa et al 2003;Hidari et al 2005). The present study demonstrates that the expression of the soluble form of the fusion protein His-GLP-N was significantly increased by decreasing the induction temperature from 37 to 268C (Fig.…”

Protein Sialylation by Sialyltransferase Involves Radiation Resistance

“…We do not know exactly why high levels of the soluble form exhibited weak sialyltransferase activity (Fig. 3); however, there has been one report in which the soluble form of ST6Gal I showed weak enzymatic activity when compared with its proform (27).…”

Converting Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) to a regioselective α2–6-sialyltransferase by saturation mutagenesis and regioselective screening

“…Other than unselective α2–6-sialylation of terminal and internal Gal or GalNAc residues by Pd2,6ST, 10, 12, 16, 17, 21 several other reported bacterial α2–6STs 26–28 including those from GT80 family have not been tested for sialylation of poly-LacNAc-type structures. While hST6Gal-I was able to be expressed in E. coli cells at a level of 0.266 mg purified enzyme per liter culture by N-terminal truncation with fusing to maltose-binding protein (MBP) 29 and the expression level was improved to 2 mg purified protein per liter culture by co-expression of multiple chaperon/foldases in the Origami2(DE3) strain, 30 the amount was still limited for large-scale synthetic purposes.…”