Improved expression and characterization of recombinant human Golgi α1, 2-mannosidase I isoforms (IA2 and IC) by Escherichia coli(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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Golgi α1,2-mannosidase I is involved in the N-linked oligosaccharide processing pathway. In this study, two truncated genes encoding for human Golgi α1,2-mannosidase I (hManIA2: amino acids 127-626 and hManIC: amino acids 118-617) were expressed in Escherichia coli to characterize the enzymes. These genes were fused to a T7 protein tag and a histidine tag at the N- and C-terminal ends, respectively, and purified using Co^<2+> affinity chromatography. The properties including optimal temperature, optimal pH, and substrate specificity of the purified enzymes were investigated by HPLC using pyridylamino (PA)-labeled oligosaccharides as substrates. The stability of hManIA2 was dependent on the presence of Ca^<2+>, which was also required for its activity. On the other hand, hManIC was stable in the absence of Ca^<2+>, even though Ca^<2+> was also effective for the activity of hManIC. While the similarity of the amino acid sequences is over 60%, hManIA2 and hManIC showed different substrate specificities particularly toward M9A and M8C.