Molecular manipulation associated with disulfide bond formation to enhance the stability of recombinant therapeutic protein

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.