Document Type: Research article
Department of Medical Biotechnology, Faculty of Medicine, Birjand University of Medical Sciences, Birjand, Iran.
Department of Medical Biotechnology, School of Advanced Technology of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Endocrinology and Metabolism Research Institute (EMRI), Tehran University of Medical Sciences, 5th Fl., Dr. Shariati Hospital, North Karegar Ave., Tehran 1411413137, Iran.
Institute of Industrial and Environmental Biotechnology (IIEB), National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box: 14965/161, Tehran, Iran
Glyco-engineering has attracted lots of interests in studies dealing with pharmacokinetics of therapeutic proteins. Based on our previous in silico studies, two sites were selected in the N-terminal gamma-carboxy glutamic acid-rich (Gla) domain of the human clotting factor IX (hFIX), to add new N-glycosylation sites. Site-directed mutagenesis was employed to conduct K22N and R37N substitutions and introduce new N-glycosylation sites in the mature hFIX. The expression efficiencies of the mutants, in parallel with the wild type hFIX (hFIXwt), were assessed in suspension adapted Chinese hamster ovary (CHO-s) cells, at transcriptional, translational and post-translational levels. The transcription levels of the both N-glycosylation mutants were significantly lower than that of the hFIXwt. In contrast, at protein level, the two hFIX mutants showed higher expression. Occurrence of hyper-glycosylation was only confirmed in the case of the hFIXR37N mutant, which decreased the clotting activity. The higher expression of the hFIX mutants, at protein level was evidenced, which could be attributed to higher protein stability, via omitting certain protease cleavage sites. The coagulation activity decline in the hyper-glycosylated hFIXR37N mutant, is probably due to the interference of the new N-glycan with protein-protein interactions in the coagulation cascade.