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Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases.
Appl Environ Microbiol. 1993 Aug; 59(8):2442-8.AE

Abstract

Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.

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Authors+Show Affiliations

Almond BD
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Dean DH
No affiliation info available

MeSH

Bacillus thuringiensisBacillus thuringiensis ToxinsBacterial ProteinsBacterial ToxinsEndopeptidasesEndotoxinsGenes, BacterialHemolysin ProteinsMutagenesis, Site-DirectedRestriction MappingThermolysinTrypsin

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8368834

Citation

Almond, B D., and D H. Dean. "Structural Stability of Bacillus Thuringiensis Delta-endotoxin Homolog-scanning Mutants Determined By Susceptibility to Proteases." Applied and Environmental Microbiology, vol. 59, no. 8, 1993, pp. 2442-8.
Almond BD, Dean DH. Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases. Appl Environ Microbiol. 1993;59(8):2442-8.
Almond, B. D., & Dean, D. H. (1993). Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases. Applied and Environmental Microbiology, 59(8), 2442-8.
Almond BD, Dean DH. Structural Stability of Bacillus Thuringiensis Delta-endotoxin Homolog-scanning Mutants Determined By Susceptibility to Proteases. Appl Environ Microbiol. 1993;59(8):2442-8. PubMed PMID: 8368834.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases. AU - Almond,B D, AU - Dean,D H, PY - 1993/8/1/pubmed PY - 2000/3/11/medline PY - 1993/8/1/entrez SP - 2442 EP - 8 JF - Applied and environmental microbiology JO - Appl Environ Microbiol VL - 59 IS - 8 N2 - Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays. Three groups of mutants could be identified. Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter). These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases. Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin. In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin. Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type. In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures. The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints. Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins. Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins. SN - 0099-2240 UR - https://www.unboundmedicine.com/medline/citation/8368834/Structural_stability_of_Bacillus_thuringiensis_delta_endotoxin_homolog_scanning_mutants_determined_by_susceptibility_to_proteases_ L2 - https://journals.asm.org/doi/10.1128/aem.59.8.2442-2448.1993?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -