title: Cell-free protein-based enzyme discovery and protein-ligand interaction study.
abstract: Cell-free expression-based screening is sometimes more suitable than cell-based assays for enzyme discovery. The advantage of cell-free systems for expression of toxic, poorly expressed, or insoluble proteins has already been well documented. Cell-free methods can advantageously replace cell-based ones when screening has to be performed on cell lysates prepared from harvested cells, for instance, when dealing with protein-ligand interactions particularly when the latter is hydrophobic. From our experience, cell-free extracts efficient in both transcription and translation can be prepared from potentially any microorganism. Here we present a general method for preparation of cell-free extracts from prokaryotic and eukaryotic cells, selection of the best systems, and optimized conditions for specific protein expression. The method allows to select proteins for their ability to bind a selected target, to identify the inhibitors of such binding, or to identify novel enzymatic activities.
authors.affiliation: Protéus, Parc Georges Besse, Nîmes, France.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Guillemer
authors.forename: Cécile
authors.initials: C
authors.lastname: Persillon
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Masson
authors.forename: Gilles
authors.initials: G
authors.lastname: Ravot
mesh_terms: D001483:Base Sequence; D002457:Cell Extracts; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014242:Trichoderma / Q000166:cytology
chemical_list: D002457:Cell Extracts; D004798:Enzymes; D008024:Ligands; D011506:Proteins