Library methods for structural biology of challenging proteins and their complexes

•Random library or directed evolution strategies in structural biology.•Mutation and screening for soluble protein expression.•Review of mutation and screening strategies with case studies

Genetic engineering of constructs to improve solubility or stability is a common approach, but it is often unclear how to obtain improvements. When the domain composition of a target is poorly understood, or if there are insufficient structure data to guide sited directed mutagenesis, long iterative phases of subcloning or mutation and expression often prove unsuccessful despite much effort. Random library approaches can offer a solution to this problem and involve construction of large libraries of construct variants that are analysed via screens or selections for the desired phenotype. Huge improvements in construct behaviour can be achieved rapidly with no requirement for prior knowledge of the target. Here we review the development of these experimental strategies and recent successes.