ArticleScientific reports2017
High-throughput mutagenesis using a two-fragment PCR approach.
Article in Scientific reports, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed.
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- Distinct stabilization of the human T cell leukemia virus type 1 immature Gag lattice.Nature structural & molecular biology · 2025Article
- A universal cannabinoid CB1 and CB2 receptor TR-FRET kinetic ligand-binding assay.Frontiers in pharmacology · 2025Article
- Agonist efficacy at the βFrontiers in pharmacology · 2025Article
- Determinants in the HTLV-1 Capsid Major Homology Region that are Critical for Virus Particle Assembly.Journal of molecular biology · 2024Article
- Structural basis of chiral wrap and T-segment capture byProceedings of the National Academy of Sciences of the United States of America · 2024Article
- Discovery of isoquinoline sulfonamides as allosteric gyrase inhibitors with activity against fluoroquinolone-resistant bacteria.Nature chemistry · 2024Article
- Molecular determinants of ligand efficacy and potency in GPCR signaling.Science (New York, N.Y.) · 2023Article
- Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- Unraveling allostery within the angiotensin II type 1 receptor for GαScience signaling · 2023Article
- Article
- Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.Nature catalysis · 2023Article
- Human Immunodeficiency Virus Type 2 Capsid Protein Mutagenesis Reveals Amino Acid Residues Important for Virus Particle Assembly.Journal of molecular biology · 2022Article
- The pocketome of G-protein-coupled receptors reveals previously untargeted allosteric sites.Nature communications · 2022Article
- A simplified Gibson assembly method for site directed mutagenesis by re-use of standard, and entirely complementary, mutagenesis primers.BMC biotechnology · 2022Article
- Functional solubilization of the βiScience · 2021Article
- GPCR activation mechanisms across classes and macro/microscales.Nature structural & molecular biology · 2021Article
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.
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