Production of the ¹⁴C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coliin small-scale batch fermentations

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce 14C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-14 C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L-1. Concentrations of 12.6 g 14C-labeled glycerol L-1 (1 % v/v) resulted in the production of 17.1 mg 14C-Cry1Ab L-1 cultivation medium. 14C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total 14C originated from Cry1Ab. In the presence of 2.04 MBq 14C-labeled carbon sources, a specific activity of up to 268 Bq mg -1 14 C-Cry1Ab was obtained. A more than three-fold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that 14C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple 14C-labeled carbon source. It also provides a general strategy to produce 14C-labeled proteins useful for soil metabolic studies