Directed Enzyme Evolution: Screening and Selection Methods (Methods in Molecular Biology, 230)

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

Directed evolution, the application of evolutionary design to enzyme engineering, requires effective screening strategies to isolate those proteins that perform a desired function from the libraries generated by the techniques. In Directed Enzyme Evolution: Screening and Selection Methods, seasoned practitioners from many leading laboratories describe their leading and readily reproducible screening strategies for isolating useful clones. These techniques have been optimized for sensitivity, high throughput, and robustness, and are of proven utility for directed evolution purposes. The assays presented use a variety of techniques, including genetic complementation, microtiter plates, solid-phase screens with colorimetric substrates, and flow cytometric screens. There are also representative examples of how phage libraries may be interrogated for enzymatic activity. Each protocol contains detailed step-by-step instructions and many notes on how best to deal with the problems that may occur. An accompanying volume, Directed Evolution Library Creation: Methods and Protocols (ISBN 1-58829-285-1), describes readily reproducible methods for the creation of mutated DNA molecules and DNA libraries.
Taken together, Directed Enzyme Evolution: Screening and Selection Methods and Directed Evolution Library Creation: Methods and Protocols capture for newcomers and more experienced investigators alike all the key methods for using directed protein evolution to better understand protein structure-function relationships, to discover new enzymes and therapeutic proteins, and to design new assays suitable for specific applications.