Searching Sequence Space to Engineer Proteins: Exponential Ensemble
Mutagenesis
Delagrave S., Youvan D. C.
Massachusetts Institute of Technology, Department of Chemistry, Cambridge 02139.
We describe an efficient method for generating combinatorial libraries with a high
percentage of unique and functional mutants. Combinatorial libraries have been
successfully used in the past to express ensembles of mutant proteins in which all
possible amino acids are encoded at a few positions in the sequence. However, as more
positions are mutagenized the proportion of functional mutants is expected to decrease
exponentially. Small groups of residues were randomized in parallel to identify, at each
altered position, amino acids which lead to functional proteins. By using optimized
nucleotide mixtures deduced from the sequences selected from the random libraries, we have
simultaneously altered 16 sites in a model pigment binding protein: approximately one
percent of the observed mutants were functional. Mathematical formalization and
extrapolation of our experimental data suggests that a 107-fold increase in the
throughput of functional mutants has been obtained relative to the expected frequency from
a random combinatorial library. Exponential ensemble mutagenesis should be advantageous in
cases where many residues must be changed simultaneously to achieve a specific engineering
goal, as in the combinatorial mutagenesis of phage displayed antibodies. With the enhanced
functional mutant frequencies obtained by this method, entire proteins could be
mutagenized combinatorially.