Article

Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis.

Department of Biochemistry and Molecular Biology, University of Georgia, 413 Biological Sciences Building, 120 Cedar Street, Athens, GA 30602, USA.
Insect Biochemistry and Molecular Biology (impact factor: 3.25). 11/2003; 33(10):999-1010. DOI:10.1016/S0965-1748(03)00114-0 pp.999-1010
Source: PubMed

ABSTRACT The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides. Manduca sexta, the tobacco hornworm, is the model insect for B. thuringiensis studies. Although brush border vesicles prepared from larval M. sexta midgut have been used in numerous mode-of-action studies of B. thuringiensis toxins, their protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome. Sub-proteomes were also established for B. thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins. Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches. Results generally did not produce matches to M. sexta proteins, but did match proteins of other Lepidoptera. Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N. Aminopeptidase N was the only GPI-anchored protein identified. Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots.

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Keywords

accurate protein identifications
 
B. thuringiensis Cry1Ac binding proteins
 
B. thuringiensis toxins
 
Bacillus thuringiensis
 
bind Cry1Ac
 
Cry1Ac binding proteins
 
crystal proteins
 
GPI-anchored proteins
 
larval M. sexta midgut
 
M. sexta midgut
 
M. sexta proteins
 
Manduca sexta
 
match proteins
 
membrane alkaline phosphatase
 
midgut brush border proteome
 
novel proteins
 
Peptide mass fingerprints
 
protein components
 
two-dimensional gel electrophoresis
 
western blots
 

Rebecca J McNall