Design of temperature-sensitive mutants solely from amino acid sequence

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-activating-sequence-Gal4 system.

Fig. 1.

Active mutants show a Ts phenotype when expressed at an appropriate level. Phenotypes of M97F (A), L50D (B), and WT CcdB (C) as a function of inducer concentration are shown. A Ts phenotype is seen in the presence of 0.06% arabinose in each case. M97F and L50D are expressed in the E. coli strain Top10 and WT in XL1Blue.

Fig. 2.

Verification of the Ts phenotype of Gal4 mutants at 21°C, 30°C, and 37°C on SC-UAH plates. Plates are sectioned into six cultures. (A) From section 1 to section 6 (clockwise) are WT, inactive (L62P, E76G double mutant), F68K, F68R, F68E, and F68P grown at 21°C, 30°C, and 37°C. F68K and F68R are mild Ts mutants, and F68E and F68P are strong Ts mutants. (B) From section 1 to section 6 (clockwise) are WT, inactive (L62P, E76G double mutant), F68Q, F68D, L69P, and L70P grown at 21°C, 30°C, and 37°C. F68Q, L69P, and L70P are mild Ts mutants, and F68D is a strong Ts mutant.