Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats

• Published in: Analytical biochemistry Vol. 412; no. 2; pp. 210 - 216
• Main Authors: Pershad, Kritika, Sullivan, Mark A., Kay, Brian K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-05-2011
• Subjects: Affinity-capture; Alkaline Phosphatase; Alkaline phosphatase fusions; Animals; Bacteriophages - genetics; Blotting, Western; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Gene Expression; Genetic Techniques; Genetic Vectors - genetics; Humans; Indicators and Reagents; Peptide Library; Phagemid vector; Recombinant Fusion Proteins - metabolism; Religation; Reproducibility of Results; Restriction enzymes; Single-Chain Antibodies - isolation & purification; Zebrafish; Phagemid vector; Affinity-capture; Restriction enzymes; Religation; Alkaline phosphatase fusions
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.02.006

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.