title: Pooling for improved screening of combinatorial libraries for directed evolution.
abstract: Following diversity generation in combinatorial protein engineering, a significant amount of effort is expended in screening the library for improved variants. Pooling, or combining multiple cells into the same assay well when screening, is a means to increase throughput and screen a larger portion of the library with less time and effort. We have developed and validated a Monte Carlo simulation model of pooling and used it to screen a library of beta-galactosidase mutants randomized in the active site to increase their activity toward fucosides. Here, we show that our model can successfully predict the number of highly improved mutants obtained via pooling and that pooling does increase the number of good mutants obtained. In unpooled conditions, we found a total of three mutants with higher activity toward p-nitrophenyl-beta-D-fucoside than that of the wild-type beta-galactosidase, whereas when pooling 10 cells per well we found a total of approximately 10 improved mutants. In addition, the number of "supermutants", those with the highest activity increase, was also higher when pooling was used. Pooling is a useful tool for increasing the efficiency of screening combinatorial protein engineering libraries.
authors.affiliation: School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 30332-0100, USA.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Polizzi
authors.forename: Monal
authors.initials: M
authors.lastname: Parikh
authors.forename: Cody U
authors.initials: CU
authors.lastname: Spencer
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsumura
authors.forename: Jay H
authors.initials: JH
authors.lastname: Lee
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Realff
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Bommarius
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D019020:Directed Molecular Evolution / Q000379:methods*; D006027:Glycosides / Q000737:chemistry; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics*
chemical_list: D006027:Glycosides; D019151:Peptide Library; C014125:4-nitrophenylfucoside; D001616:beta-Galactosidase