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

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Hemophilia Research

Background:

• Hemophilia B treatment relies on recombinant coagulation factor IX (rFIX) administration.
• Enhancing rFIX activity is crucial for therapeutic efficacy.
• The gamma-carboxylation process, influenced by propeptide residues, is key to FIX function.

Purpose of the Study:

• To investigate the contribution of specific propeptide residues to FIX gamma-carboxylation and activity.
• To engineer rFIX variants with enhanced activity through targeted protein modification.
• To optimize the gamma-carboxylic recognition site (γ-CRS) for improved γ-carboxylase interaction.

Main Methods:

• Site-directed mutagenesis was used to substitute propeptide residues (-14, -13, -12) in rFIX.
• Recombinant FIX variants were expressed in Drosophila S2 cells.
• Total FIX protein expression, active FIX levels, and specific FIX activity were quantified.

Main Results:

• Simultaneous substitution of residues at positions -12, -13, and -14 significantly increased FIX protein expression and activity.
• Enhanced rFIX activity was confirmed by improved functional, fully gamma-carboxylated FIX species.
• Barium citrate adsorption enriched for these highly active FIX variants.

Conclusions:

• Increasing the net charge and hydrophobicity of the FIX γ-CRS region enhances gamma-carboxylation.
• This modification leads to increased expression of fully active FIX.
• Targeted protein engineering of the γ-CRS offers a promising strategy for developing more effective rFIX therapies.