Design of temperature-sensitive mutants solely from amino acid sequence

被引:62
作者
Chakshusmathi, G
Mondal, K
Lakshmi, GS
Singh, G
Roy, A
Ch, RB
Madhusudhanan, S
Varadarajan, R [1 ]
机构
[1] Indian Inst Sci, Mol Biophys Unit, Bangalore 560012, Karnataka, India
[2] Jawaharlal Nehru Ctr Adv Sci Res, Chem Biol Unit, Bangalore 560004, Karnataka, India
关键词
D O I
10.1073/pnas.0402222101
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Temperature-sensitive (Ts) mutants area 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 (it) 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 upstrearn-activatingsequence-Gal4 system.
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页码:7925 / 7930
页数:6
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