Enhanced Toxicity of Purified Bacillus thuringiensis Cry1Ac-endotoxin Singh Vivek Kumar1,4, Nain Vikrant2, Sharma Priyanka1, Rao K R S Sambasiva4, Birah Ananta3, Gupta G P3, Kumar P. A.1,5,* 1National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012, India 2Gautam Buddha University, Greater Noida, India 3Division of Entomology, Indian Agricultural Research Institute, New Delhi-110012, India 4Acharya Nagarjuna University, Guntur, India 5Indian Institute of Rice research, Rajendra Nagar, Hyderabad-500030, India *For Correspondence-polumetla@hotmail.com
Online published on 16 December, 2015. Abstract Use of Cry1Ac δ-endotoxin of Bacillus thuringiensis (Bt) has revolutionized crop protection especially in cotton. Large quantities of Cry1Ac protein are needed for several basic studies. Heterologous expression in E. coli facilitates production of large quantities of recombinant Cry1Ac protein. However, earlier studies reported that E. coli expressed recombinant protein leads to large variations in LC50 values. Here we report a method of active Cry1Ac δ-endotoxin purification that is simple, robust and exhibits higher toxicity with consistent results in insect bioassays. In this protocol proteolysis separates completely folded Cry1Ac δ-endotoxin molecules (active toxin) from nonnative protein folding forms and other host proteins, followed by extraction of active Cry1Ac toxin by micro filtration. Insect bioassay of purified Cry1Ac toxin with Helicoverpa armigera showed 125 times enhanced toxicity (LC50 40 pg/cm2) as compared to the earlier reports (LC50 ng/cm2) 4.5 to 3500 ng/cm2. These results provide a new prospective in determination of baseline susceptibility, monitoring resistance development and utilization of its potential. Top Keywords δ-endotoxin, Helicoverpa armegera, CHAPS, PVDF membrane, PAGE, protein purification, microfiltration. Top |