title: Conceptual and methodological advances in cell-free directed evolution.
abstract: Although cell-free directed evolution methods have been used to engineer proteins for nearly two decades, selections on more complex phenotypes have largely remained in the domain of cell-based engineering approaches. Here, we review recent conceptual advances that now enable in vitro display of multimeric proteins, integral membrane proteins, and proteins with an expanded amino acid repertoire. Additionally, we discuss methodological improvements that have enhanced the accessibility, efficiency, and robustness of cell-free approaches. Coupling these advances with the in vitro advantages of creating exceptionally large libraries and precisely controlling all experimental conditions, cell-free directed evolution is poised to contribute significantly to our understanding and engineering of more complex protein phenotypes.
authors.affiliation: Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Igor
authors.initials: I
authors.lastname: Dodevski
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: George C
authors.initials: GC
authors.lastname: Markou
authors.affiliation: Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA; Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: csarkar@umn.edu.
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D019020:Directed Molecular Evolution; D008565:Membrane Proteins / Q000737:chemistry; D010641:Phenotype; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D008565:Membrane Proteins; D011506:Proteins