Enhancement of Purified Human Colon Cancer-Specific Parasporal Toxin from Bacillus thuringiensis-LDC-501.

Abstract

Parasporal inclusion protein of Bacillus thuringiensis-LDC-501 (Bt-LDC-501) exhibits selective cytocidal action towards human colon cancer cells. The yield of this parasporal protein was minimum in the normal culture. In order to increase the yield of protein from Bt-LDC-501 various agro-based cost-efficient nutrient sources such as corn steep liquor (CSL), sesame oil cake extract (SOC), groundnut oil cake extract (GOC), neem oil cake extract (NOC), rice bran extract (RB), wheat bran extract (WB), red gram hull extract (RGH), green gram hull extract (GGH), black gram hull extract (BGH), Mysore gram hull extract (MGH), and maize flour waste extract (MFW) were screened. Statistical experimental designs such as Plackett-Burman design (PBD) and response surface methodology (RSM) were the tools employed for the optimization of medium. Groundnut cake extract (GOC) served as a potential carbon and nitrogen source, as it induced twofold higher production of parasporal protein. Among the optimized seven media components KH2PO4, K2HPO4, GOC, NaCl, MgSO4, MnSO4, and FeSO4, the concentrations of GOC, NaCl, and MgSO4 have significant effect on parasporin production as well as cytotoxicity against colon cancer cell line, HCT-116. Bt-LDC-501 was found to produce 0.88mg/ml of parasporal protein in optimized medium. In the un-optimized medium, the yield was 0.23mg/ml only. This indicated that there was 382% of increase in the production of Parasporal protein. Parasporin protein with the molecular weight of 27kDa has been purified with the purification fold of 27.1. It showed a LC50 value of 0.91 and 1.21µg/ml against colorectal cancer cell lines such as HCT-116 and HCT-15, respectively. Purified parasporin exhibited stable cytocidal activity between pH 4.0 and 9.0 at room temperature. The present study revealed that the quantity and quality of media composition were necessary for eliciting cytocidal activity against human colon cancer and the importance of alternate cost-effective production of clinically significant parasporin. Moreover, this is the first report regarding optimization of media components for parasporal protein production from Bt.