Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences 1 1Edited by J. Wells

Abstract

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-®delity DNA shuf¯ing and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuf¯ing con®rmed important features of the recombination process: rapid ®xation and accumulation of bene®cial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The ®ve to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described.# 1997 Academic Press Limited

A quantitative, high-throughput screen for protein stability

“…Combinatorial and directed-evolution methods have proven to be promising techniques for designing proteins of novel structure and function (27,28). These methods systematically generate a large number of sequences and it is imperative to be able to detect successful de novo designs from a large background of unfolded polypeptides.…”

Perspective: Transposable Elements, Parasitic Dna, and Genome Evolution

“…Although simple DNA base substitutions are well suited for the generation, diversification, and optimization of local protein space (Maeshiro and Kimura 1998), a hierarchy of natural mutational events is required for the rapid generation of protein diversity. Experimental evolution (Moore et al 1997;Zhang et al 1997;Crameri et al 1998) and computer simulation experiments (Bogarad and Deem 1999) showed that sequence shuffling has the potential to improve protein function significantly better than does point mutation alone. Because they are uniquely competent to reshuffle DNA sequences, TEs and viruses are important generators of the more complex types of mutations in the mutational hierarchy.…”

Design by Directed Evolution

“…20 For N sequences with M total mutations, the probability of generating the sequence containing all of the mutations (the rarest sequence) by random recombination is 1/N M . This becomes small very quickly when multiple sequences (or many mutations) are recombined.…”