Consensus-based engineering of protein stability: From intrabodies to thermostable enzymes

Publisher Summary This chapter elaborates the consensus-based engineering of protein stability. Protein stability is an experimental metric of the information encoded in a protein sequence. This approach has allowed to engineer hyperstable immunoglobulin domains that can be expressed as intrabodies. Its application by a number of groups to a diverse set of proteins has demonstrated this procedure to be the most reliable strategy for the rational generation of stabilizing mutations. The ability to generate synthetic enzymatically active proteins from consensus sequences emphasizes the statistical nature of natural protein sequences as products of neutral drift and selective pressure. Stabilization is not the only objective for which the approach is useful, and others could include the selection of residues for nondisruptive destabilizing mutations to reduce the lifetime of proteins, or to generate proteins that can be used to screen for second-site revertants in evolutionary engineering strategies. It is found that the final steps of mutagenesis, characterization, and combination of successful residues are specific for each experimental system at hand and should be straightforward.