biostudies-literatureUnknown101(21)Chakshusmathi GTemperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Putative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four prediProceedings of the National Academy of Sciences of the United States of America7925-30https://www.ebi.ac.uk/biostudies/studies/S-EPMC419533biostudies-literatureDesign of temperature-sensitive mutants solely from amino acid sequence.PMC419533Singh GCh RBRoy AChakshusmathi GLakshmi GSMadhusudhanan SMondal KVaradarajan RfalseDesign of temperature-sensitive mutants solely from amino acid sequence.Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Putative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predi2004-01-01T00:00:00Z2004 May2025-05-29T16:00:56.193Z2025-05-29T16:00:56.193ZS-EPMC4195331514836310.1073/pnas.0402222101