title: Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).
abstract: Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS.
authors.affiliation: The Florey Institute of Neuroscience and Mental Health and The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Yong
authors.forename: D J
authors.initials: DJ
authors.lastname: Scott
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012967:Sodium Dodecyl Sulfate
keywords: cellular high-throughput encapsulation solubilization and screening (CHESS); directed evolution; enzyme stabilization; green fluorescent protein (GFP); protein engineering; protein stabilization