Rapid sequence scanning mutagenesis Using in Silico oligo design and the megaprimer PCR of whole plasmid method (MegaWHOP)

Abstract

A wide variety of random- and site-directed mutagenesis techniques have been developed to investigate the structure-function relationship in proteins and intergenic regions like promoter sequences. Similar techniques can be employed to optimize protein properties like enantioselectivity, substrate specificity, and stability in a directed evolution approach. Due to the tremendous genetic diversity that is created by common random-mutagenesis methods, directed evolution techniques usually require the time-consuming and cumbersome screening of large numbers of variants. A gene-scanning saturation-mutagenesis approach represents one efficient way to limit the screening effort by reducing the created genetic diversity. In structure/function studies often a similar method, e.g., alanine- or arginine-scanning mutagenesis, is used to probe the role of specific amino acids in a protein. Here, we present a standardized mutagenesis strategy that can speed up the process of scanning whole proteins for structure/function studies and, furthermore, allows for the fast and efficient generation of gene-scanning saturation-mutagenesis libraries to be used in the directed evolution of enzyme functions and properties. The described method uses automated computer-assisted oligonucleotide design, and a two-step PCR-mutagenesis protocol to amplify site-specifically mutated circular plasmids that can be directly transformed in Escherichia coli expression strains. © 2010 Springer Science+Business Media, LLC.