Assaying phage-borne peptides by phage capture on fibrinogen or streptavidin.
There is no simple and efficient method for assaying phage isolated from libraries without having to resort to PEG purification of the phage, or to the biotinylation or other labelling of the target molecule. We report here a method for producing 'bifunctional' phage that express two types of peptide; one peptide, fused to pVIII, will bind to immobilized fibrinogen, allowing capture of the phage out of culture supernatants; this allows the other peptide, fused to pIII or pVIII to be assayed by simple ELISA. This system has also been developed for the capture of phage bearing a streptavidin-binding peptide. The bifunctional phage are produced by bacterial cells bearing a plasmid that expresses pVIII fused either to the fibrinogen-binding peptide or to the streptavidin-binding one. Thus, when these cells are infected with a phage clone or pool to be assayed, phage will be produced whose 'capture-peptide' is produced from the plasmid and whose 'assay-peptide' is produced from the phage genome. We show here that, by this method, bifunctional phage can be produced that will bind to immobilized streptavidin or fibrinogen.
Bonnycastle
L L
LL
Institute of Molecular Biology and Biochemistry, Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
Brown
K L
KL
Tang
J
J
Scott
J K
JK
D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D017911:Inoviridae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000138:chemical synthesis; D011485:Protein Binding; D019809:Streptavidin
D001426:Bacterial Proteins; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D005340:Fibrinogen; D019809:Streptavidin