Effect of propeptide amino acid substitution in γ-carboxylation, activity and expression of recombinant human...

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre-propeptide sequences. The propeptide is connected to γ-carboxylase enzyme through the γ-carboxylase recognition site for the direct γ-carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ-carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ-carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT-hFIX-M14 expression cassette, containing cDNA of hFIX with substituted -14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4-fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD-14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated 2-fold enhanced recovery in the S2-expressing hFIXD-14A relative to that expressed native hFIX. These results show that changing -14 residues leads to a decrease in the binding affinity to substrate, increase in γ-carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515-520, 2018.