Molecular Genetic Manipulation of Truncated Cry1C Protein Synthesis in Bacillus thuringiensis To Improve Stability and Yield

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Abstract
Cry1 protoxins of Bacillus thuringiensisare insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by usingcyt1A promoters to drive expression of the 5′ half ofcry1C that included in the construct the 5′cry3A STAB-SD mRNA stabilizing sequence and the 3′ stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3′ transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only thecyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC50] = 13.2 μg/ml) was lower than that of full-length Cry1C (LC50 = 1.8 μg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp.kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

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