Gene Cloning, Expression, and Purification of Kunitz Trypsin Inhibitor from Glycine max Using Halo Tag.

Soybean Kunitz Trypsin Inhibitor (SKTI) is one of the most extensively studied protease inhibitors, with applications in pest management, medicine, the food processing industry, and the leather industry. In this study, SKTI was cloned into the pFN29A Flexi vector containing a barnase gene. Genomic DNA was isolated from tender soybean leaves, and SKTI was amplified by PCR to obtain a 671 bp product. After cloning, an internal 380 bp sequence was amplified using specific primers to confirm that the cloned sequence was a functional SKTI, as non-functional SKTI genes also exist in Glycine max. The amplified PCR product, containing an AsiSI site at the 5' end and a PmeI site at the 3' end, was cloned into the pFN29A vector. The resulting colonies were screened by colony PCR, and the insert sequence was confirmed by Sanger sequencing. The recombinant protein, containing a His-tag, Halo-tag, and a TEV protease cleavage site, was expressed in Escherichia coli BL21 cells. Maximum expression was achieved 5 h after induction with 0.5 mM IPTG at 37 °C. The expressed SKTI was purified using affinity chromatography on HaloLink resin, and the bound SKTI was cleaved with HaloTEV protease to obtain pure SKTI. The purified inhibitor effectively inhibited bovine trypsin, with an IC₅₀ of 0.6 ± 0.003 µg/µl, yielding 1.6 mg per gram of bacterial pellet. The 24 kDa inhibitor remained stable up to a temperature of 50 °C. Kinetic analysis revealed that recombinant SKTI competitively inhibits trypsin, with a Kᵢ value of 14 µM.