Expression and purification of cytokine receptor homology domain of human granulocyte-colony stimulating factor receptor in Escherichia coli.

In an attempt to generate a stable non-glycosylated cytokine receptor homology (CRH) domain (Tyr(97)-Ala(309)) of human granulocyte-colony stimulating factor (G-CSF) receptor, two free cysteines in the CRH domain were converted to serine by site-directed mutagenesis. Taking advantage of the tight regulation for the expression of T7 RNA polymerase, the mutated CRH domain was successfully expressed in Escherichia coli (E. coli) with a pelB signal sequence at its NH2-terminus and with a His tag at its COOH-terminus. The processed and secreted CRH domain after solubilization and in vitro refolding retained G-CSP binding activity, and its yield (similar to 40 mu g/30 ml culture) was more than 100-fold higher than that of the mouse CRH domain expressed by the MalE fusion system in E. coli.