Thünen Institute (vTI) for Biodiversity, Federal Research Institute for Rural Areas, Forestry and Fisheries, Bundesallee 50, 38116 Braunschweig, Germany.
Appl Microbiol Biotechnol. 2012 Oct;96(1):221-9. doi: 10.1007/s00253-012-4299-2. Epub 2012 Jul 31.
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 (14)C-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 (14)C-labeled glycerol L(-1) (1 % v/v) resulted in the production of 17.1 mg (14)C-Cry1Ab L(-1) cultivation medium. (14)C 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 (14)C originated from Cry1Ab. In the presence of 2.04 MBq (14)C-labeled carbon sources, a specific activity of up to 268 Bq mg(-1) (14)C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that (14)C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple (14)C-labeled carbon source. It also provides a general strategy to produce (14)C-labeled proteins useful for soil metabolic studies.
苏云金芽孢杆菌自然产生的杀虫晶体蛋白是转基因作物表达的主要重组性状。它们在种植期间和之后释放到土壤中。本研究的目的是生产 14C 标记的 Cry1Ab 蛋白，用于其环境风险评估范围内的土壤代谢研究。Cry1Ab 由大肠杆菌 HB101 pMP 在 200 毫升液体分批培养发酵中作为原毒素合成，并在胰蛋白酶消化后从包涵体中纯化。对于培养，U-14C-甘油是主要的碳源。当培养肉汤中总有机碳的初始量低于 6.4mg C L-1 时，包涵体更小，Cry1Ab 的产量更低。12.6g 14C 标记的甘油 L-1（1%v/v）的浓度导致 17.1mg 14C-Cry1Ab L-1 培养介质的产生。14C 质量平衡表明，约 50%的标记通过呼吸损失，20%留在生长介质中，而剩余的活性与生物量相关。根据生产批次的不同，0.01%至 0.05%的总 14C 来自 Cry1Ab。在存在 2.04MBq 14C 标记碳源的情况下，获得高达 268Bqmg-1（14C-Cry1Ab）的比活性。在存在 4.63MBq 和延长的培养期 144h 的情况下，获得了比活性高三倍以上的比活性。本研究表明，在简单 14C 标记碳源存在的情况下，可从大肠杆菌的分批发酵中获得 14C 标记的 Cry1Ab。它还提供了一种生产用于土壤代谢研究的 14C 标记蛋白的一般策略。
