Open AccessIsolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site‐directed mutagenesis system
Author(s) -
Meza Roberto,
NuñezValdez MariaEugenia,
Sanchez Jorge,
Bravo Alejandra
Publication year - 1996
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1996.tb08597.x
Subject(s) - mutagenesis , mutant , bacillus thuringiensis , site directed mutagenesis , serine protease , biochemistry , biology , salt bridge , arginine , aspartic acid , directed mutagenesis , serine , mutation , histidine , amino acid , protease , chemistry , genetics , gene , bacteria , enzyme
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 α‐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.