In vitro evolution of glutathione S-transferase using a plasmid display system based on the GAL4 DNA-binding domain

Enzyme characteristics, such as thermal stability and catalytic activity, can be improved in a targeted manner. However, the screening of target mutants is time-consuming and requires highly experimental downstream efforts. Here, we describe a simple strategy based on plasmid display and limited proteolysis for the rapid and easy screening of highly stable and active mutants from a library. When glutathione S-transferase was used as a model enzyme, the resulting mutants obtained in the first round of screening were approximately two- to sevenfold more thermostable than the wild-type enzyme at 50 °C, with similar enzyme activity. This methodology is therefore powerful for the in vitro enrichment and screening of thermostable and active mutants. It can reduce downstream experimental effort and can create a high-quality library using relatively simple steps.