Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis.
A fully automated peptide synthesizer has been constructed that is capable of the simultaneous synthesis of up to 36 individual peptides and the synthesis of equimolar peptide mixtures. The instrument consists of an array of reaction vessels, a series of solenoid valves to control liquid flow, and a Zymark robot to deliver solvents and reagents; all components are computer controlled and coordinated. Equimolar peptide mixtures are obtained by algorithms that automate the mixing and distribution of peptide-resin particles. This technology was used to synthesize a library of 361 peptides, generated by randomizing two critical binding residues of a 10-mer epitope known to bind an anti-human immuno-deficiency virus gp120 monoclonal antibody. Each critical residue was substituted with 19 amino acids consisting of all the natural amino acids except cysteine. The library was synthesized as 19 pools, each containing 19 peptides. Each pool was screened in a solution-phase competition ELISA assay. The 12 most inhibitory peptides in the library were isolated by a rapid affinity-selection method and were identified by mass spectrometry and amino acid analysis. The binding properties of these 12 selected peptides were verified by synthesis and assay of the individual peptides. The two critical residues investigated were found to contribute independently to antibody binding.
Zuckermann
R N
RN
Kerr
J M
JM
Siani
M A
MA
Banville
S C
SC
Santi
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification; D051379:Mice / Q000276:immunology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000276:immunology; D012371:Robotics
D000911:Antibodies, Monoclonal; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D009842:Oligopeptides