Improved activity and expression of recombinant human factor IX by propeptide engineering

Abstract

Purpose The main therapeutic strategy for Hemophilia B patients involves the administration of recombinant coagulation factors IX (rFIX). Although there are various approaches to increasing the activity of rFIX, targeted protein engineering of specific residues could result in increased rFIX activity through enhanced gamma-carboxylation. Specific amino acids in the propeptide sequence of vitamin K-dependent proteins are known to play a role in the interaction with the enzyme gamma-carboxylase. The net hydrophobicity and charge of the gamma-carboxylic recognition site (gamma-CRS) region in the propeptide are important determinants of gamma-carboxylase binding. So the contribution of individual gamma-CRS residues to the expression of fully gamma-carboxylated and active FIX was studied. Methods Propeptide residues at positions -14, -13, or - 12 were substituted for equivalent prothrombin amino acids by SEOing PCR. The recombinant FIX variants were transfected and stably expressed in Drosophila S2 cells, and the expression of both total FIX protein and active FIX was assessed. Results While overall the substitutions resulted in an increase of both total FIX protein expression as well as an increase in the portion of active FIX, the highest increase in FIX protein expression, FIX activity, and specific FIX activity was observed following the simultaneous substitution of residues at positions -12, -13, and - 14. The enhanced rFIX activity was further confirmed by enrichment for functional, fully gamma-carboxylated rFIX species via barium citrate adsorption. Conclusion Our findings indicate that by increasing both the net charge and the net hydrophobicity of the FIX gamma-CRS region, the expression of fully gamma-carboxylated and as such active FIX is enhanced.