title: [A reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human blood type B→O conversion].
abstract: This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.
authors.affiliation: Department of Blood Molecular Biology, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Gao
authors.forename: Su-Bo
authors.initials: SB
authors.lastname: Li
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Bao
authors.forename: Ying-Xia
authors.initials: YX
authors.lastname: Tan
authors.forename: Ling-Yan
authors.initials: LY
authors.lastname: Wang
authors.forename: Si-Hu
authors.initials: SH
authors.lastname: Jin
authors.forename: Ying-Li
authors.initials: YL
authors.lastname: Wang
authors.forename: Shou-Ping
authors.initials: SP
authors.lastname: Ji
authors.forename: Feng
authors.initials: F
authors.lastname: Gong
mesh_terms: D000017:ABO Blood-Group System / Q000276:immunology*; D001441:Bacteroides fragilis / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis; D000519:alpha-Galactosidase / Q000096:biosynthesis*
chemical_list: D000017:ABO Blood-Group System; D011994:Recombinant Proteins; D000519:alpha-Galactosidase