CH391L/S13/DirectedProteinEvolution

Directed evolution is a powerful method for altering the properties of biological parts and systems. Directed protein evolution employs iterative rounds of mutation and artificial selection to generate new proteins with desirable functions.

Introduction

Biological molecules have the amazing ability to rapidly evolve in response to strong selective pressure. Protein engineers exploit this evolvability to generate new and useful protein functions through successive rounds of mutation and selection. This approach is known as directed protein evolution, and it involves four basic steps:

    1. A parent protein sequence is selected.
    2. The parent sequence is mutated to generate a library of functional variants.
    3. Variants are evaluated for their ability to perform the desired function.
    4. The process is repeated until the desired function is achieved.

The parent sequence is chosen based on its perceived similarity to the desired function, and a library of functional variants is generated using one of a variety of sequences diversification techniques. High-throughput functional screens and genetic selection methods are used to identify library members with enhanced target function, and those variants are used as parent sequences in successive rounds of mutation and selection. This process is repeated until the desired function is achieved.

Library construction

After a parent sequence is chosen, a library of functional mutants must be generated. Common methods used for library construction include error-prone PCR and DNA shuffling.

Error-prone PCR is a technique for introducing random point mutations into cloned sequences, in which modifications to standard PCR conditions increase the error rate of nucleotide incorporation during amplification. Common methods for decreasing polymerase fidelity include the addition of manganese ions, an increase in the concentration of magnesium ions, and using an imbalanced ratio of dNTPs]. There are a number of commercially available kits for error-prone PCR such as the GeneMorph II random mutagenesis kit from Agilent Technologies.

DNA shuffling is technique for “in vitro homologous recombination of pools of selected mutant genes." In this method, parental sequences are fragmented by DNaseI and then reassembled by PCR. Recombination events occur as fragments anneal at regions of sufficient sequence homology. After reassembly, the chimeric sequences are amplified by PCR and cloned into an appropriate vector. Selection and screening techniques

Once a library of mutants is generated, they must be evaluated for their ability to perform the desired function (i.e. bind a specific target molecule). To do this, protein engineers employ a variety of high-throughput functional assays. Successful assays allow researchers to test a large number of functional variants while maintaining a connection between phenotype (the evolved protein function) and genotype (the DNA sequence encoding the evolved protein function).

Phage display is an assay method that allows for the identification of proteins that bind a desired target molecule. This technique has been widely used to select for and evolve antibodies for use as therapeutics. In phage display, a physical linkage between protein and DNA sequence is maintained. Related in vitro display techniques include mRNA and ribosome display methods.

Cell-based compartmentalization techniques maintain a link between phenotype and genotype that “is achieved naturally by introducing plasmid DNA encoding the protein into a cell." These methods allow millions of sequence variants to be transformed into cells, and manipulating the statistics of DNA transformation allows for each cell to contain a single vector containing a single sequence variant. These individual cells can then be isolated by growth on solid media.

List of high-throughput assays for protein function:

    1. phage display
    2. ribosome display
    3. mRNA-peptide fusion
    4. plasmid display
    5. cell-surface display
    6. genetics
    7. n-hybrid systems
    8. in vitro compartmentalization
    9. spatial address
   10. mass spectrometry

iGEM connection

The UC Davis 2012 iGEM team used directed evolution to engineer an E. coli strain that more efficiently degrades ethylene glycol than previous strains. Although not strictly a directed protein evolution project, their work demonstrates the ability of biological molecules and systems to rapidly evolve under strong selective pressure.