Abstract
A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T7 promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20mM Tris–HCl, pH 9.0, 500mM arginine, 500mM guanidine HCl, 15% glycerol, 1mM cystamine, and 5mM cysteine at 2–8°C for 40h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.
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