Zhang Lin, Moo-Young Murray, Chou C Perry
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Protein Expr Purif. 2011 Jan;75(1):28-39. doi: 10.1016/j.pep.2010.08.006. Epub 2010 Aug 16.
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.
人CD83（hCD83ext）细胞外结构域中的Cys²⁷作为一种潜在的治疗性蛋白质，被确定为分子操作的靶点。分别使用具有还原性细胞质和氧化性细胞质的两种大肠杆菌菌株BL21(DE3)和Origami B(DE3)作为表达宿主来生产Cys²⁷突变体。观察到当hCD83ext在Origami B(DE3)中表达时，Cys²⁷参与了分子内二硫键的体内形成。通过分子间二硫键的体外形成，源自Origami的蛋白质产物比源自BL21的对应产物具有更高的多聚化倾向。进行了各种分析以确定这些突变变体之间的结构差异。最重要的是，Cys²⁷突变增强了分子稳定性，因为与野生型hCD83ext相比，源自BL21或Origami的Cys²⁷突变体对降解的敏感性要低得多。这项研究突出了异常二硫键形成对治疗性蛋白质生产的影响。
