Production of the 13C/15N single-labeled insecticidal protein Cry1Ab/Ac for the assessment of metabolic fate using recombinant Escherichia coli

Abstract

Stable isotope-labeled Cry1Ab/Ac protein is necessary for the metabolic study of exogenous insecticidal protein in soil using the stable isotope labeling technique, but no recombinant expression protocols for this protein have been reported. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which obtained the safety certificate in China, was subcloned into pUC57 in this study, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM IPTG was added to these cells and cultured at 37°C for 4 h to induce the expressed protein to form inclusion bodies in the presence of M9 medium containing either [U-13C] glucose (5% 13C-enriched) or [15N] ammonium chloride (5% 15N-enriched). Then Cry inclusion bodies were dissolved in urea and purified by Ni column affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by SDS-PAGE and Western blot. The purities of 13C/15N-labeled Cry proteins were each 99%, the amounts of which were 12.6 mg/L and 8.8 mg/L. The δ 13C and δ 15N values of 13C-labeled Cry protein and 15N-labeled Cry protein were 3268.68‰ and 2854.28‰, respectively. An insecticidal test showed that the prokaryotic expression of Cry1Ab/Ac protein had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies of Cry protein in soil and the characteristics of nitrogen (N) and carbon (C) transformation. The findings will also provide a reference and basis for elucidating the environmental behaviors and ecological effects of Cry plants and expressed products.