Production of recombinant IbAr 10200 GP38his was accomplished by transient transfection of HEK293Ts (American Type Culture Collection) with the tPA-GP38his plasmid using FuGENE 6 according to the manufacturer’s instructions. Supernatants were collected 48 and 72 hours after transfection (with media replacement after 48 hours). Supernatants were clarified and mixed with protease inhibitors. Supernatants were then run over a Ni2+ HisTrap FF column (GE Healthcare) using an ÄKTA high-performance liquid chromatography system. The column was then washed with five-column volumes of binding buffer [500 mM NaCl, 10 mM imidazole, and 20 mM sodium phosphate (pH 7.4)]. Subsequently, captured GP38his was eluted off using elution buffer (binding buffer with addition of 500 mM imidazole). Fractions with the highest absorbance (at 280 nm) were then pooled and concentrated through a Centriprep 10-kDa filter device (Millipore). Final product protein concentration was determined by BCA protein assay (Thermo Fisher Scientific).

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