CH391L/S13/DirectedProteinEvolution

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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.

Library construction

There are several common techniques used to construct libraries of functional variants

Error-prone PCR

Error-prone PCR amplifies wild-type sequences using a polymerase that has an error rate of up to 2%. The PCR can be made error-prone in a number of ways, including increasing the concentration of Mg2+, adding Mn2+, or using unequal concentrations of each nucleotide. After the amplification, the mutant sequences must be cloned. The size of a library generated by error-prone PCR is limited by cloning step. Common mutations resulting from error-prone PCR are point mutations, but deletions and frameshift mutations are also possible. There are a number of commercially available kits for error-prone PCR (link to Stratagene). Similar techniques also use mutagenic primers.

Recombination of homologous sequences

Computer-guided mutagenesis

DNA shuffling

Screening techniques

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
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