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[摘要]：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. coil 0127:K63(B8) was expressed in E. coil BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the whiQ gene was identified to encode an alpha 1,2-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-beta 1,3-GaINAc-alpha-OR linkages; indicating that both the Gal and GaINAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-alpha 1,2-Gal-beta 1,3-GaINAc-alpha-OMe, on a milligram scale. (C) 2010 Elsevier Inc. All rights reserved. |
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