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Methods for the directed evolution of proteins

Key Points

  • Directed evolution is a cyclic process that alternates between gene diversification and screening for or selection of functional gene variants.

  • Library size limitations can be overcome by focusing library diversity on residues implicated by molecular structures, computational models or phylogenetic data. In cases in which there is limited information, random mutagenesis can be used to interrogate the uncertain determinants of protein function.

  • Recombination methodologies access new combinations of functional variation and can shuffle disparate genetic elements to yield new chimeric proteins.

  • Low-throughput screens can directly measure individual phenotypes and thus accurately isolate desired subpopulations. Screen throughput can be increased using indirect visible reporters that are strongly coupled to the desired phenotypes.

  • Selections isolate functional variants through selective replication schemes or physical segregation. Selections operate simultaneously on entire populations and thus offer unparalleled throughput.

Abstract

Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature.

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Figure 1: Key steps in the cycle of directed evolution.
Figure 2: Genetic recombination methods for protein evolution.
Figure 3: Screening methods for protein evolution.
Figure 4: Selection methods for protein evolution.
Figure 5: Optimal strategies for directed evolution.

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Acknowledgements

This work was supported by the US Defense Advanced Research Projects Agency grants DARPA HR0011-11-2-0003 and DARPA N66001-12-C-4207, the US National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) (grant R01 GM095501) and the Howard Hughes Medical Institute (HHMI).

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Correspondence to David R. Liu.

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PowerPoint slides

Glossary

Natural selection

A process by which individuals with the highest reproductive fitness pass on their genetic material to their offspring, thus maintaining and enriching heritable traits that are adaptive to the natural environment.

Artificial selection

(Also known as selective breeding). A process by which human intervention in the reproductive cycle imposes a selection pressure for phenotypic traits desired by the breeder.

Libraries

Diverse populations of DNA fragments that are subject to downstream screening and selection.

Library size

The number variants that are subjected to screening and selection. Library sizes are limited by molecular cloning protocols and/or by host transformation efficiency.

Focused mutagenesis

A strategy of diversification that introduces mutations at DNA regions expected to influence protein activity.

Random mutagenesis

A strategy of diversification that introduces mutations in an unbiased manner throughout the entire gene.

Mutational spectrum

The frequency of each specific type of transition and transversion. The evenness of this spectrum allows more thorough sampling of sequence space.

Transformation

The process by which a cell directly acquires a foreign DNA molecule. A number of protocols allow high-efficiency transformation of microorganisms through treatments with ionic buffers, heat shock or electroporation.

Neutral drift

A process that occurs in the presence of a purifying selection pressure to eliminate deleterious mutations. This is in contrast to genetic drift, a process by which mutations fluctuate in frequency in the absence of selection pressure.

Degenerate codons

Codons constructed with a mixed population of nucleotides at a given position, thus sampling all possible amino acids within the constructed libraries. The most popular examples are NNK and NNS (where N can be any of the four nucleotides, K can be G or T, and S can be G or C).

Epistatic interactions

Non-additive effects between mutations (for example, mutational synergy or synthetic lethality). As a result, the sequential acquisition of mutations is not always equivalent to mutational co-occurrence.

Homologous recombination

A process by which separate pieces of DNA swap genetic material, guided by the annealing of complementary DNA fragments.

Passenger mutations

(Also known as hitchhiker mutations). Unnecessary mutations that are enriched in a population owing to co-occurrence with a highly beneficial linked mutation.

Transduction

The process by which a viral vector delivers a foreign DNA molecule to a cellular host.

Evolutionary potential

The capacity of a protein to take on new functions through evolution. High thermostability allows for necessary but destabilizing mutations, and functional diversity of homologues is a demonstration of previous evolution in nature.

Surrogate substrates

Substrate analogues that are permissive of enzymatic conversion but that, upon catalysis, exhibit chemical rearrangements that lead to altered optical properties, including visible colour, relief of fluorophore quenching, shifted fluorophore excitation or emission, and downstream chemiluminescence.

Fluorescence-activated cell sorting

(FACS). A flow cytometry method in which an aqueous suspension of cells or cell-like compartments is measured for fluorescence (often at multiple wavelengths) one cell at a time and subsequently separated based on a fluorescence threshold.

Negative screen

A screening method that involves depletion of an undesired phenotype.

Positive screening

Enrichment for a desired activity such as improved kinetics, tolerance to unnatural conditions and acceptance of new substrates.

Transformation bottleneck

The efficiency at which DNA library members are transferred into the host organism, thus restricting the number of variants that can be assessed by in vivo selection and screening.

Auxotroph complementation

The ability of functional library members to resolve a metabolic defect in the host, leading to replication of DNA that encodes active library members.

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Packer, M., Liu, D. Methods for the directed evolution of proteins. Nat Rev Genet 16, 379–394 (2015). https://doi.org/10.1038/nrg3927

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