Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli
Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli
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DOI：
10.1007/s10719-005-0845-9
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发表时间：
2005-02-01
影响因子：
3
通讯作者：
Suzuki, Y
中科院分区：
文献类型：
--
作者：
Hidari, KIPJ;Horie, N;Suzuki, Y
在大肠杆菌中表达了可溶性和活性形式的重组人ST 6 Gal I。将编码缺乏膜和胞质区域的可溶形式的ST 6 Gal I的基因引入细菌表达载体pMAL-p2 X中，其与麦芽糖结合蛋白（MBP）标签框内融合。IPTG诱导过程中在13 ℃下的低温培养显著改善了细菌中表达的重组酶的溶解性和MBP标记。通过破坏细胞制备的上清液证明唾液酸转移活性的寡糖和糖蛋白，脱唾液酸胎球蛋白，表明在细菌中表达的酶是可溶的和活性的。通过阳离子交换柱和淀粉酶缀合的琼脂糖柱层析的组合，有效地纯化MBP标记的酶。纯化的重组酶即使在反应混合物中不存在去污剂的情况下也发挥酶活性。受体底物特异性的酶是略有不同，从大鼠肝ST 6 Gal I。这些观察结果表明，膜和胞质区域的ST 6 Gal I可能会影响酶的特性。纯化的重组酶用于将去唾液酸化胎球蛋白转化为再唾液酸化胎球蛋白。凝集素印迹证明，再唾液酸化胎球蛋白具有单个Neu 5Ac α 2-6残基。再唾液酸化胎球蛋白有效地阻断流感病毒株A/孟菲斯/ 1/71（H3 N2）诱导的血凝，表明蛋白质上的再唾液酸化糖链是如此活跃，以至于竞争性地抑制病毒-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ST 6 Gal I是一个有价值的工具，以调查通过碳水化合物介导的生物和病理相互作用的分子机制。
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.