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 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 phage-display 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 phage-display library both against purified ligands and more complex materials like calf serum.
Jacobsson
K
K
Swedish University of Agricultural Sciences, Uppsala, Sweden.
Frykberg
L
L
D001426:Bacterial Proteins / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D001798:Blood Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012709:Serum Albumin / Q000235:genetics; D013205:Staphylococcal Protein A / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics*
D001426:Bacterial Proteins; D001798:Blood Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D012709:Serum Albumin; D013205:Staphylococcal Protein A