A reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human blood type B→O conversion.
- Author:
Hong-Wei GAO
1
;
Su-Bo LI
;
Guo-Qiang BAO
;
Ying-Xia TAN
;
Ling-Yan WANG
;
Si-Hu JIN
;
Ying-Li WANG
;
Shou-Ping JI
;
Feng GONG
Author Information
1. Department of Blood Molecular Biology, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
- Publication Type:Journal Article
- MeSH:
ABO Blood-Group System;
immunology;
Bacteroides fragilis;
enzymology;
Cloning, Molecular;
Escherichia coli;
metabolism;
Humans;
Recombinant Proteins;
biosynthesis;
alpha-Galactosidase;
biosynthesis
- From:
Journal of Experimental Hematology
2011;19(2):503-507
- CountryChina
- Language:Chinese
-
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.
