Dataset Information

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

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.

SUBMITTER: Pershad K

PROVIDER: S-EPMC3068214 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Pershad Kritika K Sullivan Mark A MA Kay Brian K BK

Analytical biochemistry 20110301 2

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 gener ...[more]

PMID: 21315061

Similar Datasets

Project description:Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure