title: Improved expression and characterization of recombinant human Golgi α1,2-mannosidase I isoforms (IA2 and IC) by Escherichia coli.
abstract: 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.
authors: International Center for Biotechnology, Osaka University, 2–1 Yamada-oka, Suita, Osaka 565–0871, Japan.
authors: Junko
authors: J
authors: Isoyama-Tanaka
authors: Koji
authors: K
authors: Dohi
authors: Ryo
authors: R
authors: Misaki
authors: Kazuhito
authors: K
authors: Fujiyama
mesh_terms: D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D020033:Protein Isoforms; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002118:Calcium