Production of Universal Group O Red Blood Cells by Alpha-N-Acetylgalactosaminidase Enzyme Expressed in Pichia pastoris

Enzymatic removal of blood groups antigens A and B is an efficient method for production of universal red blood cells. In this research, an -N-acetylgalactosaminidase (NAGA) enzyme was expressed in Pichia pastoris for digestion of the A blood antigen. DNA sequence of the gene NAGA, originally expressed in Elizabethkingia meningosepticum (NAGA-EM), was ordered for optimization and synthesis. It was then expressed in P. pastoris (KM71H and GS115 strains). Expression of the recombinant NAGA was evaluated by dot blot, SDS-PAGE, and Western blotting. The activity of the enzyme was measured using a synthetic substrate in addition to the conversion of group A red blood cells to the O cells. Expression of NAGA-EM with an apparent molecular mass of 55kDa was verified by dot blot, SDS-PAGE and Western blot analysis. The maximum enzyme activity in the supernatant of KM71H was higher than that in the GS115 (250 vs. 200 U/ml). Treated group A RBCs did not react with the anti-A antiserum or with the sera from individuals with blood groups B and O. The results of this study indicated that NAGA-EM is an efficient enzyme for production of universal O blood cells.