High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries.
The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.
González-Techera
A
A
Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, and Departamento de Inmunobiología, Facultad de Medicina, UDELAR, Montevideo, Uruguay.
Umpiérrez-Failache
M
M
Cardozo
S
S
Obal
G
G
Pritsch
O
O
Last
J A
JA
Gee
S J
SJ
Hammock
B D
BD
González-Sapienza
G
G
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000915:Antibody Affinity; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000276:immunology; D008024:Ligands; D008958:Models, Molecular; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase