Paths to Directed Protein Evolution; An Experimental Exploration of Protein Sequence Space

摘要

Directed protein evolution has emerged as a powerful approach for the engineering of protein function for practical applications. Traits that can be altered by directed protein evolution include enzyme specificity; stability to denaturation, the introduction of allostery (in other words the regulation of function by non-substrate small molecules), binding specificity etc. So far the most established way for the 'laboratory' evolution " of a desired protein function is iterative low level mutagenesis and screening. Briefly, a library of mutants is generated under conditions that result in 1-2 amino acid substitutions per protein. The mutated gene pool is expressed in microorganisms giving rise to a "library" of clones that are screened for improved function, usually by colony or microtiter well plate assays. Clones that produce improved or evolved function are isolated and resubjected to mutagenesis and screening. In vitro recombination by DNA shuffling can also be employed to accelerate the generation of mutants that contain multiple beneficial substitutions.