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

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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∘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 α 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. Published in 2005.

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Authors and Affiliations

  1. Department of Biochemistry, School of Pharmaceutical Sciences, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada, Shizuoka City, Shizuoka, 422-8526, Japan

    Kazuya I. P. J. Hidari, Daisei Miyamoto, Takashi Suzuki & Yasuo Suzuki

  2. Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka City, Shizuoka, 422-8529, Japan

    Nobuhiro Horie, Takeomi Murata & Taiichi Usui

  3. Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City, Shizuoka, 422-8526, Japan

    Yasuo Suzuki

  4. Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City, Shizuoka, 422-8526, Japan

    Kazuya I. P. J. Hidari

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  1. Kazuya I. P. J. Hidari
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  2. Nobuhiro Horie
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  3. Takeomi Murata
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  4. Daisei Miyamoto
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  5. Takashi Suzuki
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  7. Yasuo Suzuki
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Correspondence to Yasuo Suzuki.

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Hidari, K.I.P.J., Horie, N., Murata, T. et al. Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli . Glycoconj J 22, 1–11 (2005). https://doi.org/10.1007/s10719-005-0845-9

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  • DOI: https://doi.org/10.1007/s10719-005-0845-9

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