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Appl. Environ. Microbiol. doi:10.1128/AEM.00752-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Bacillus thuringiensis serovar thompsoni HD542 Crystal Proteins: Solubilization, Activation, and Insecticidal Activity

Department of Plant Physiology and Molecular Biology, University of Plovdiv, 24 Tzar Assen Street, 4000 Plovdiv, Bulgaria; Department of Biochemistry, Wageningen University and Research Centre, P.O. Box 8128, 6700 ET Wageningen, The Netherlands; Busines Unit Bioscience, Plant Research International B.V, Wageningen University and Research Centre, P.O. Box 16, 6700 AA Wageningen, The Netherlands

^* To whom correspondence should be addressed. Email: naimov0{at}uni-plovdiv.bg.

	Abstract

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40 kDa accompanying protein is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper we describe the characterization of the Cry15Aa and 40 kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40 kDa protein to fragments too small to be detected by SDS-PAGE, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris brassicae were susceptible to crystals and pre-solubilization of the crystals enhanced activity to P. brassicae. Activity for all three species was enhanced by pre-incubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40 kDa crystal protein showed no activity in the insects tested, nor did its addition or co-expression in E. coli increase the activity of Cry15 in insecticidal and hemolytic assays.