WbiQ 的特性：大肠杆菌 O127:K63(B8) 的 α1,2-岩藻糖基转移酶，以及 H 型 3 血型抗原的合成。
Characterization of WbiQ: An α1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen.
机构信息
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
出版信息
Biochem Biophys Res Commun. 2010 Nov 12;402(2):190-5. doi: 10.1016/j.bbrc.2010.08.087. Epub 2010 Aug 27.
Abstract
Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an α1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-β1,3-GalNAc-α-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe, on a milligram scale.
摘要
大肠杆菌 O127:K63(B8) 由于其 O-抗原重复单元结构而具有高的人血红细胞 H（O）活性。在这项工作中，来自大肠杆菌 O127:K63(B8) 的 wbiQ 基因在大肠杆菌 BL21(DE3) 中表达，并作为含有 N 端 GST 亲和标签的融合蛋白进行纯化。使用 GST-WbiQ 融合蛋白，通过放射性测定、薄层层析分析以及使用质谱和 NMR 光谱法确认产物形成，鉴定 wbiQ 基因编码一种 α1,2-岩藻糖基转移酶。融合酶（GST-WbiQ）的最佳 pH 范围为 6.5 至 7.5，并且不需要二价金属存在即可具有酶活性。WbiQ 显示出严格的底物特异性，仅对含有 Gal-β1,3-GalNAc-α-OR 键的受体具有活性；表明 Gal 和 GalNAc 残基对酶活性都是至关重要的。此外，WbiQ 被用于在毫克规模上制备 H 型 3 血型抗原，Fuc-α1,2-Gal-β1,3-GalNAc-α-OMe。
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