title: In vitro evolution of glutathione S-transferase using a plasmid display system based on the GAL4 DNA-binding domain.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Chungnam National University, 99 Daehak-ro (St), Yuseong-gu, Daejeon, 305-764, Korea. biochoi@cnu.ac.kr
authors.forename: Yoo Seong
authors.initials: YS
authors.lastname: Choi
authors.forename: Young Je
authors.initials: YJ
authors.lastname: Yoo
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D015723:Gene Library; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D010957:Plasmids; D055550:Protein Stability
chemical_list: D004247:DNA; D005982:Glutathione Transferase