Phage display of a biologically active Bacillus thuringiensis toxin

• Published in: Applied and Environmental Microbiology 1998-08, Vol.64 (8), p.2995-3003
• Main Authors: Kasman, L.M. (University of Georgia, Athens, GA.), Lukowiak, A.A, Garczynski, S.F, McNall, R.J, Youngman, P, Adang, M.J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Bacillus Phages - genetics; Bacillus thuringiensis; Bacillus thuringiensis - genetics; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proteins - toxicity; BACTERIAL TOXINS; BACTERIOFAGOS; BACTERIOPHAGE; Bacteriophage lambda - genetics; BACTERIOPHAGES; Biological and medical sciences; Biotechnology; COAT PROTEINS; ELISA; Endotoxins - chemistry; Endotoxins - genetics; Endotoxins - metabolism; Endotoxins - toxicity; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Genetic technics; Genetic Vectors; HELIOTHIS VIRESCENS; Hemolysin Proteins; IMMUNOBLOTTING; INSECTICIDAL PROPERTIES; INSECTICIDAS; INSECTICIDE; INSECTICIDES; Invertebrate Microbiology; Methods. Procedures. Technologies; MICROBIAL PROTEINS; Miscellaneous; Molecular biology; Molecular Sequence Data; Peptide Library; PROTEINAS MICROBIANAS; PROTEINAS RECOMBINANTES; PROTEINE MICROBIENNE; PROTEINE RECOMBINANTE; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - toxicity; RECOMBINANT PROTEINS; Synthetic digonucleotides and genes. Sequencing; TEST ELISA; TOXINAS BACTERIANAS; TOXINE BACTERIENNE; WESTERN BLOTTING
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.8.2995-3003.1998

Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning