Production of Universal Group O Red Blood Cells by Alpha-N-Acetylgalactosaminidase Enzyme Expressed in Pichia pastoris
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Enzymatic removal of blood groups antigens A and B is an efficient method for production of universal red blood cells. In this research, an α-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 55 kDa 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.
KeywordsAntigen A α-N-acetylgalactosaminidase Codon optimization Pichia pastoris NAGA-EM
This manuscript was extracted from the dissertation that was funded by a research grant from Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine for M.Sc. degree of Medical Biotechnology (Thesis No. 14). The lab work was approved as research Project No. 931563, and carried out the Department of Medical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. The authors would like to appreciate support of the Transfusion Center, Mashhad, Iran for providing anonymous aliquots of blood samples of groups used in this project.
Compliance with Ethical Standards
Conflict of interest
All the authors declare that they have no conflict of interest.
This project was approved by the local ethics committee (Code: IR.MUMS.FM.Rec.1394.228) at Mashad University of Medical Sciences, Mashhad, Iran.