Gene VIII-Based, Phage-Display Vectors for Selection Against Complex Mixtures of Ligands
Selection of shotgun phage-display libraries against complex mixtures of components, such as cells or sera, may result in a high number of nonspecifically binding phage. Consequently, correct interactions may be difficult to identify. To enable discrimination between faithful and nonspecific interac...
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| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
1998-02-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/98242rr01 |
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| Summary: | Selection of shotgun phage-display libraries against complex mixtures of components, such as cells or sera, may result in a high number of nonspecifically binding phage. Consequently, correct interactions may be difficult to identify. To enable discrimination between faithful and nonspecific interactions, a set of eight different gene VIII-based, phage-display vectors were constructed. All vectors contain a “universal” screening tag positioned in such a way that it is only expressed when the inserted DNA encodes an open reading frame, which corrects a shift of reading frames in the vector. A Staphylococcus aureus shotgun phagedisplay library was made in a stoichiometric mixture of all vectors. After affinity-selection against IgG, one vector completely outcompeted the others. This vector contains the promoter and signal sequence from the gene encoding staphylococcal protein A and one suppressible stop codon immediately upstream of gene VIII. An increase in the frequency of clones expressing the affinity tag in all pannings correlated with selection for ligand-binding clones. This enables detection of putatively correct clones after selection of a shotgun phagedisplay library both against purified ligands and more complex materials like calf serum. |
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| ISSN: | 0736-6205 1940-9818 |