Abstract
Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies.
Similar content being viewed by others
Author information
Authors and Affiliations
Additional information
Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996
Rights and permissions
About this article
Cite this article
Gustafson, M., Clayton, R., Lavrik, P. et al. Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli . Appl Microbiol Biotechnol 47, 255–261 (1997). https://doi.org/10.1007/s002530050923
Issue Date:
DOI: https://doi.org/10.1007/s002530050923