Recursive ensemble mutagenesis.

We have developed a generally applicable experimental procedure to find functional proteins that are many mutational steps from wild type. Optimization algorithms, which are typically used to search for solutions to certain combinatorial problems, have been adapted to the problem of searching the 'sequence space' of proteins. Many of the steps normally performed by a digital computer are embodied in this new molecular genetics technique, termed recursive ensemble mutagenesis (REM). REM uses information gained from previous iterations of combinatorial cassette mutagenesis (CCM) to search sequence space more efficiently. We have used REM to simultaneously mutate six amino acid residues in a model protein. As compared to conventional CCM, one iteration of REM yielded a 30-fold increase in the frequency of 'positive' mutants. Since a multiplicative factor of similar magnitude is expected for the mutagenesis of additional sets of six residues, performing REM on 18 sites is expected to yield an exponential (30,000-fold) increase in the throughput of positive mutants as compared to random [NN(G,C)]18 mutagenesis.