Phage display of a biologically active Bacillus thuringiensis toxin.
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.
Department of Entomology, University of Georgia, Athens, Georgia 30602, USA.
L M
LM
Kasman
A A
AA
Lukowiak
S F
SF
Garczynski
R J
RJ
McNall
P
P
Youngman
M J
MJ
Adang
D000595:Amino Acid Sequence; D000818:Animals; D017103:Bacillus Phages / Q000235:genetics*; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins; D010582:Bacteriophage lambda / Q000235:genetics; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D006460:Hemolysin Proteins; D015151:Immunoblotting; D007306:Insecticides; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis