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Design of temperature-sensitive mutants solely from amino acid sequence

Chakshusmathi, Ghadiyaram and Mondal, Kajari and Lakshmi, Santosh G and Singh, Guramrit and Roy, Ankita and Babu, Ravindra Ch and Madhusudhanan, S and Varadarajan, Raghavan (2004) Design of temperature-sensitive mutants solely from amino acid sequence. In: Proceedings of the National Academy of Sciences, 101 (21). pp. 7925-7930.

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Abstract

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 predicted buried sites. By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites. The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68, 69, and 70. No Ts mutants of either protein have been previously reported. Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activatingsequence –Gal4 system.

Item Type: Journal Article
Additional Information: The copyright belongs to National Academy of Sciences.
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 02 May 2006
Last Modified: 19 Sep 2010 04:26
URI: http://eprints.iisc.ernet.in/id/eprint/6511

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