Fig. 2

Purity of recombinant cytokine preparations and folding of recombinant aa2-152 IL-38. A. To assess the purity of the recombinant protein preparations used in this study, 1 µg of each sample (aa2-152 IL-38 with C-terminal His tag, aa2-152 IL-38 in storage buffer and in PBS, aa20-152 IL-38:Fc-KIH fusion protein, Fc-KIH IgG1 control, left gel; aa3-152 IL-38, middle gel; IL-36Ra, sIL-1Ra, icIL-1Ra1, right gel) were fractionated by SDS-PAGE and stained with Coomassie Blue. Based on calculated molecular weights (MW), expected sizes are: 18 kDa for aa2-152His IL-38 and icIL-1Ra1, 17 kDa for aa2-152 IL-38, aa3-152 IL-38, IL-36Ra and sIL-1Ra1, 28 kDa and 50 kDa for IL-38:Fc-KIH, 28 kDa for Fc-KIH IgG1 control. The size (kDa) of MW markers is indicated on the left. Storage buffer: 30 mM Hepes pH 7, 500mM NaCl, 5% glycerol, 1 mM β-ME. Fc ctrl: Fc-KIH IgG1. B. Folding of recombinant IL-38 aa2-152 was analyzed by circular dichroism. The observed percentage of β-strands was approximately 40%. CD, circular dichroism; Mdeg, measured ellipticity. C. X-ray diffraction crystal structure of the human IL-38 protein without the initiator methionine and with a Cys-to-Ser mutation at position 2 (PDB: 5BOW). β-strands (orange) represent 40.1% of the sequence, while the remaining regions (dark blue) are structured as coils and short α-helices