24] Selecting and evolving functional proteins in vitro by ribosome display

Abstract

Publisher Summary In vivo systems have a number of limitations. One important restriction is that the library size is limited by the transformation efficiency, a step that all of these techniques have in common. Another limitation of in vivo -based selection methods becomes apparent if a diversification step must be included. It requires either repeatedly switching between in vivo selection and in vitro diversification or the use of mutator strains. The former approach is quite laborious, as it makes a new library generation and large-scale transformation necessary for each cycle of sequence diversification and selection. The latter approach may have the disadvantage that possible candidate molecules may be removed from the library by mutations generated either in the host genome or in the plasmid regions important for expression or replication. All these limitations can be simultaneously overcome in ribosome display. Ribosome display is an in vitro technology for the simultaneous selection and evolution of proteins from diverse libraries. Because no transformation is necessary, large libraries can be prepared and applied for selection. Diversification is conveniently introduced in this method, thereby making evolutionary approaches easily accessible. Ribosome display was first applied to short peptides and has subsequently been improved to work with folded proteins. This chapter describes in detail the methodology and the nature of the improvements and provides the information necessary to perform ribosome display for the selection and evolution of functional proteins, using either an E. coli or a rabbit reticulocyte translation system.