A method for directed evolution and functional cloning of enzymes.
A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
H
H
Pedersen
S
S
Hölder
D P
DP
Sutherlin
U
U
Schwitter
D S
DS
King
P G
PG
Schultz
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D019020:Directed Molecular Evolution; D004798:Enzymes / Q000235:genetics*; D008969:Molecular Sequence Data
D017931:DNA Primers; D004798:Enzymes