GENE EXPRESSION, PURIFICATION, AND CYTOTOXIC ANALYSIS OF RECOMBINANT FUSION PROTEIN OF CAJANUS CAJAN TRYPSIN INHIBITOR WITH EPINECIDIN-1

Abstract Trypsin inhibitors, also called serpins are the largest class of protease inhibitors having antagonistic properties to catalytic proteases. In this study, a 528bp Cajanus cajan trypsin inhibitor gene (CcTI) fused with a 63bp epinecidin-1 peptide (GFIFHIIKGLFHAGKMIHGLV), an anti-microbial peptide (AMP) from Epinephelus coioides, was cloned in pET-29a(+) expression vector and transformed in E. coli host system (Top10 and Bl21 cells) to produce rCcTI-epi-1 protein. Molecular cloning and chromatographic techniques were employed to maximize the production of biologically important trypsin inhibitory protein with a potent defensive role in therapeutics. The recombinant inhibitor-peptide complex (rCcTI-epi-1) was expressed in soluble lysate as well as inclusion bodies at reducing IPTG condition (0.5mM) with increasing the incubation time (24hrs). Contrary to insoluble protein aggregates, the expression levels of rCcTI-epi-1 protein were high in soluble lysate extracts. The 21kDa recombinant trypsin inhibitor-epinecidin-1 protein was purified by affinity chromatography at pH 7.5 with high purification yield but low purity of rCcTI-epi-1 protein using 250mM imidazole while elution with 500mM imidazole showed low yield but high on purity rCcTI-epi-1 protein. The rCcTI-epi-1 protein exhibited high microbial inhibition against Escherichia coli at a dose of 18μg/ml, whereas, the antimicrobial effect of rCcTI-epi-1 against Staphylococcus aureus was low (36μg/ml), mainly exerted by epinecidin-1 AMP peptide. The IC50 values of the cytotoxic effect of rCcTI-epi-1 protein against MDA-MB-231, MCF-7, A549, and HepG2 cell lines were 15.86μg/ml, 17.33μg/ml, 18.17μg/ml, and 20.25μg/ml, respectively. Taken together, our study investigate comprehensive invitro therapeutic potential of rCcTI-epi-1 protein against different carcinomas, uncovering the clinical potency of rCcTI-epi-1 as a targeted agent for cancer treatment in the future.