Purification Process And Activity Determination Of Human Recombinant SPINK6 Protein

Human serine proteinase inhibitor, Kazal-type6(SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6has been shown to selectively inhibit kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor.Here, we report the successful generation of tagless SPINK6using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and Trypsin-affinity chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. The yield of rhSPINK6can reach12.5mg/L culture supernatant. After two-step purification, the recombinant protein can reach95%purity and approximate40%recovery.Pichia pastoris-derived recombinant human SPINK6(rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase14(KLK14)(Ki=0.16nM) than previously reported Escherichia coli-derived rhSPINK6(Ki=0.5nM). This protein also exhibited moderate inhibition of bovine trypsin (Ki=33nM), while previous E. coli-derived rhSPINK6did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.