Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system.
A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.
Meza
R
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Instituto de Biotecnologícal Universidad Nacional Autónoma de México, Cuernavaca, Mor., Mexico.
Nuñez-Valdez
M E
ME
Sanchez
J
J
Bravo
A
A
D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular; D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005420:Flavoproteins / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017956:Photoreceptor Cells, Invertebrate; D010940:Plant Proteins / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000502:physiology; D043562:Receptors, G-Protein-Coupled; D012492:Salts / Q000032:analysis; D055672:Static Electricity
D001426:Bacterial Proteins; D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005420:Flavoproteins; D010940:Plant Proteins; D043562:Receptors, G-Protein-Coupled; D012492:Salts; C119828:cry protein, Drosophila; D001224:Aspartic Acid; D001120:Arginine