ArticleMethods (San Diego, Calif.)2012
Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.
Article in Methods (San Diego, Calif.), 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 54 citations in OpenAlex.
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- Heat-sterilizable antibody mimics designed on the cold shock protein scaffold from hyperthermophile Thermotoga maritima.Protein science : a publication of the Protein Society · 2025Article
- Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.Nature chemical biology · 2024Article
- Sequence-independent, site-specific incorporation of chemical modifications to generate light-activated plasmids.Chemical science · 2023Article
- Epivolve: A Protocol for Site-Directed Antibodies.Methods in molecular biology (Clifton, N.J.) · 2023Article
- High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.Nature communications · 2022Article
- Resurfaced ZIKV EDIII nanoparticle immunogens elicit neutralizing and protective responses in vivo.Cell chemical biology · 2022Article
- Engineering pH-Sensitive Single-Domain Antibodies.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.Nucleic acids research · 2021Article
- Article
- Evolving a Peptide: Library Platforms and Diversification Strategies.International journal of molecular sciences · 2019Review
- Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.International journal of molecular sciences · 2019Review
- Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.eLife · 2019Article
- Surface Loops in a Single SH2 Domain Are Capable of Encoding the Spectrum of Specificity of the SH2 Family.Molecular & cellular proteomics : MCP · 2019Article
- Identification of variant HIV envelope proteins with enhanced affinities for precursors to anti-gp41 broadly neutralizing antibodies.PloS one · 2019Article
- Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3.The Journal of biological chemistry · 2018Article
- Generating FN3-Based Affinity Reagents Through Phage Display.Current protocols in chemical biology · 2018Article
- Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity.Journal of visualized experiments : JoVE · 2018Article
- Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
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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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.