Characterization Of Wbiq: An Alpha 1,2-Fucosyltransferase From Escherichia Coli O127:K63(B8), And Synthesis Of H-Type 3 Blood Group Antigen

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 0127: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 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.