Production and Purification of Recombinant Kunitz Domain Proteins

The recombinant protein rSmKI-1 and its fragments, Kunitz (KI) domain (N-terminal Arg²²–Thr⁸²) and C-terminal tail (C-terminal Gli⁷⁹–Glu¹⁴⁶), were produced as previously described (10 (link)). Briefly, plasmids containing the sequence for rSmKI-1 or KI-domain or C-terminal tail from SmKI-1 were transformed into E. coli Rosetta™ (Merck KGaA, Darmstadt, Germany) competent cells. Cells transformed were cultured in selective medium and gene expression was induced by 1 mM isopropylthiogalactoside (IPTG). After induction, the bacterial cells were harvested and recombinant proteins were recovered as inclusion bodies and solubilized. Each protein was purified by affinity chromatography on a Ni-Sepharose column (Hitrap chelating 5 mL) using an AKTA prime Plus chromatography system (GE Healthcare, São Paulo, Brazil) according to the manufacturer’s protocol. Fractions containing proteins used in this study were determined through SDS/PAGE-20% and then, dialyzed against PBS pH 7.0. The recombinant proteins were quantified using the BCA kit (Pierce, Waltham, MA, USA). To evaluate the amount of endotoxin present, the samples were submitted to Limulus Amebocyte Lysate QCL-1000™ (Lonza) assay. Protein samples show less than 1 endotoxin unit (EU)/mg.