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), and Zebrafish Core Facility-EMRI, Tehran University of Medical Sciences, Tehran, 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), Tehran, Iran.
Glyco-engineering has attracted lots of interest in studies dealing with the 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 both N-glycosylation mutants were significantly lower than that of the hFIXwt. In contrast, at the protein level, the two hFIX mutants showed higher expression. The 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.