Production of a novel hyper-glycosylated human coagulation factor IX in HEK293 cells, using a Glyco-engineering Approach

Ghasemi, ّFahimeh; Zomorodipour, Alireza; Karkhane, Ali Asghar

Production of a novel hyper-glycosylated human coagulation factor IX in HEK293 cells, using a Glyco-engineering Approach

Document Type : Original Research

Authors

ّFahimeh Ghasemi ¹

Alireza Zomorodipour ²

Ali Asghar Karkhane ³

¹ 1. Department of Medical Biotechnology, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran.2. Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran.

² Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology

³ Institute of Industrial and Environmental Biotechnology (IIEB), National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

Abstract

Hyper-glycosylation is an approach to introduce new N-glycosylation consensus sequence(s) (َAsn-Xxx-Ser/Thr three-peptide) into a protein primary amino acid sequences by site-directed mutagenesis which is followed by the attachment of a new glycan to the Asn residue located within the three-peptide sequence. Hyper-glycosylation has attracted lots of interest especially in the protein therapeutics industry. The attached glycan may improve the pharmacokinetic properties of the hyper-glycosylated priteins and increase their half-life in the bloodstream. In the current study, a new N-glycosylation site was introduced into N-terminal Gla domain of hFIX. Arg³⁷ position of mature hFIX was targeted to be converted into Asn residue by site-directed mutagenesis using overlap extension PCR. Recombinant expression plasmids for native and mutant hFIX were constructed. The expression of the recombinant wild-type and mutant hFIX was analyzed in mammalian HEK293 cells using gradient SDS-PAGE and western blotting analysis. The results indicated in higher molecular weight for R37N mutant in compared with the native protein. The glycan attachment to R37N mutant was further confirmed by PNGase digestion and western blotting.

Keywords

Recombinant human coagulation factor IX

Hyper-glycosylation

HEK293 cells

20.1001.1.23222115.1399.11.3.3.6

Subjects

Pharmaceutical Biotechnology

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