Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents.
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http://dx.doi.org/10.1016/j.ymeth.2012.08.008 | DOI Listing |
Nat Commun
October 2024
Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, US National Institutes of Health, Department of Health and Human Services, 111 TW Alexander Drive, Research Triangle Park, NC, 27709, USA.
Chem Sci
November 2023
Department of Chemistry, University of Oxford Mansfield Road OX1 3TA Oxford UK
Plasmids are ubiquitous in biology, where they are used to study gene-function relationships and intricate molecular networks, and hold potential as therapeutic devices. Developing methods to control their function will advance their application in research and may also expedite their translation to clinical settings. Light is an attractive stimulus to conditionally regulate plasmid expression as it is non-invasive, and its properties such as wavelength, intensity, and duration can be adjusted to minimise cellular toxicity and increase penetration.
View Article and Find Full Text PDFMethods Mol Biol
September 2023
Tango Biosciences, Chicago, IL, USA.
An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 10 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev.
View Article and Find Full Text PDFMethods Mol Biol
September 2023
Center of Molecular Immunology, La Habana, Cuba.
The current chapter focuses on the use of filamentous phages to display and modify biologically active cytokines, with special emphasis on directed evolution of novel variants showing improved receptor binding. Cytokines are essential protein mediators involved in cell-to-cell communication. Their functional importance and the complexity of their interactions with multichain receptors make cytokine engineering a promising tool for the discovery and optimization of therapeutic molecules.
View Article and Find Full Text PDFNucleic Acids Res
December 2022
Genome Integrity & Structural Biology Laboratory, Princeton University, Princeton, NJ 08544, USA.
Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication.
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