Hidari Kazuya I P J, Horie Nobuhiro, Murata Takeomi, Miyamoto Daisei, Suzuki Takashi, Usui Taiichi, Suzuki Yasuo
Department of Biochemistry, School of Pharmaceutical Sciences, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, University of Shizuoka.
Glycoconj J. 2005 Feb;22(1-2):1-11. doi: 10.1007/s10719-005-0845-9.
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.
重组人ST6Gal I的一种可溶且有活性的形式在大肠杆菌中表达。编码缺失膜区和胞质区的ST6Gal I可溶形式的基因被导入细菌表达载体pMAL-p2X中，与麦芽糖结合蛋白（MBP）标签框内融合。在IPTG诱导期间于13℃进行低温培养显著提高了细菌中表达的重组酶的溶解度和MBP标签化程度。通过细胞破碎制备的上清液对寡糖和糖蛋白去唾液酸胎球蛋白均表现出唾液酸转移活性，表明在细菌中表达的酶是可溶且有活性的。通过阳离子交换柱和淀粉酶偶联琼脂糖柱层析相结合的方法有效地纯化了带有MBP标签的酶。纯化的重组酶即使在反应混合物中没有去污剂的情况下也能发挥酶活性。该酶的受体底物特异性与大鼠肝脏ST6Gal I的略有不同。这些观察结果表明ST6Gal I的膜区和胞质区可能会影响该酶的性质。将纯化的重组酶用于将去唾液酸化的胎球蛋白转化为再唾液酸化的胎球蛋白。凝集素印迹表明再唾液酸化的胎球蛋白含有一个Neu5Acα2-6残基。再唾液酸化的胎球蛋白有效地阻断了甲型流感病毒株A/孟菲斯/1/71（H3N2）诱导的血凝，表明蛋白质上再唾液酸化的糖链具有很强的活性，能够竞争性抑制病毒-受体相互作用。总之，使用我们的细菌表达系统获得的可溶性重组ST6Gal I是研究通过碳水化合物介导的生物和病理相互作用分子机制的有价值工具。
