ArticleAnalytical biochemistry2011
Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats.
Article in Analytical biochemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
What it found
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Who cites it
9 citing papers in PubMed, 13 citations in OpenAlex.
- Engineering pH-Sensitive Single-Domain Antibodies.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Article
- A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1.International journal of molecular sciences · 2018Article
- Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase.PloS one · 2016Article
- Article
- Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.PloS one · 2014Article
- Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.Journal of molecular biology · 2012Article
- Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.Methods (San Diego, Calif.) · 2012Article
- Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase.New biotechnology · 2012Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
Abstract
Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments.
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Registered trials
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.