Molecular manipulation associated with disulfide bond formation to enhance the stability of recombinant therapeutic
Lin Zhang1, Murray Moo-Young, C Perry Chou
1Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Abstract:
Cys²⁷ in the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein, was identified as a target for molecular manipulation. Two Escherichia coli strains of BL21(DE3) and Origami B(DE3), respectively, with a reducing and an oxidative cytoplasm were used as the expression host to produce the Cys²⁷ mutants. It was observed that Cys²⁷ was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3). The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of intermolecular disulfide bonds. Various analyses were conducted to identify the structural differences among these mutant variants. Most importantly, molecular stability was enhanced by the Cys²⁷ mutations since the Cys²⁷ mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext. This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins.
Related Concept Videos
Protein Modifications in the RER
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Protein Folding
Protein Structure Is Critical to Its Biological Function
Proteins perform a wide range of biological functions such as catalyzing chemical reactions, providing...
Protein Folding
Tagging and Fusion Proteins
Preparation and Reactions of Thiols
Production of Pharmaceuticals


