Structural perturbation and compensation by directed evolution at physiological temperature leads to thermostabilization of β-lactamase

被引:56
作者
Hecky, J [1 ]
Müller, KM [1 ]
机构
[1] Univ Freiburg, Inst Biol 3, D-79104 Freiburg, Germany
关键词
D O I
10.1021/bi0501885
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The choice of protein for use in technical and medical applications is limited by stability issues, making understanding and engineering of stability key. Here, enzyme destabilization by truncation was combined with directed evolution to create stable variants of TEM-1 beta-lactamase. This enzyme was chosen because of its implication in prodrug activation therapy, pathogen resistance to lactam antibiotics, and reporter enzyme bioassays. Removal of five N-terminal residues generated a mutant which did not confer antibiotic resistance at 37 degrees C. Accordingly, the half-life time in vitro was only 7 s at 40 degrees C. However, three cycles comprising random mutagenesis, DNA shuffling, and metabolic selection at 37 degrees C yielded mutants providing resistance levels significantly higher than that of the wild type. These mutants demonstrated increased thermoactivity and thermostability in time-resolved kinetics at various temperatures. Chemical denaturation revealed improved thermodynamic stabilities of a three-state unfolding pathway exceeding wild-type construct stability. Elongation of one optimized deletion mutant to full length increased its stability even further. Compared to that of the wild type, the temperature optimum was shifted from 35 to 50 degrees C, and the beginning of heat inactivation increased by 20 degrees C while full activity at low temperatures was maintained. We attribute these effects mainly to two independently acting boundary interface residue exchanges (M 182T and A224V). Structural perturbation by terminal truncation, evolutionary compensation at physiological temperatures, and elongation is an efficient way to analyze and improve thermostability without the need for high-temperature selection, structural information, or homologous proteins.
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页码:12640 / 12654
页数:15
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