Abstract Background and Objectives The shortage of different ABO blood groups is a permanent problem that many blood transfusion centers encounter. The conversion of blood group A and B to group O is a solution to this problem. In this research we aimed to clone and express the α-galactosidase enzyme gene to convert the blood group B to O.
Materials and Methods In this experimental study, the α-galacosidase gene from coffee bean was codon-optimized and cloned into the pBHA plasmid. After replication in E. coli, plasmid was cut with BamH1 and Nco1 restriction enzymes and the α-galacosidase gene was purified by gel electrophoresis. The gene was then cloned into the expression vector, pET-20b. The expression construct was introduced into E. coli Rosetta 2 (DE3) and the expression of the enzyme was induced by IPTG. The presence of protein production was investigated using SDS-PAGE and Western blotting. Protein function test was also evaluated by the ability of B cells to convert to O cells.
Results The presence of the recombinant protein was confirmed by SDS-PAGE and Western blot assay. The purified protein could partially convert blood group B RBCs into group O as detected by decreasing the agglutination strength with B antiserum from 4+ to less than 1+.
Conclusions The production of eukaryotic proteins in a prokaryotic host is a major step in mass production. Optimization of different expression conditions is essential to achieve sufficient amount of enzyme.
Hadad deilami A, Salehi M, Shahabi M. Cloning and expression a-galactosidase in order to convert B to O blood group. Sci J Iran Blood Transfus Organ 2022; 19 (4) :301-312 URL: http://bloodjournal.ir/article-1-1450-en.html