ABSTRACT
The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide-beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-beta-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μkappa) of two murine B-cell lymphoma cell lines (antigen unknown).
ABSTRACT
Selectide Technology is a random synthetic combinatorial library method in which millions of random compounds are screened in parallel for their ability to bind to a tagged macromolecular target. The library consists of millions of beads and each individual bead expresses a unique chemical compound such as a peptide. In the standard enzyme-linked colorimetric detection scheme, the positive bead which turns color is isolated for microsequencing. In this paper, a dual color detection scheme using two sequential orthogonal probes is described. This dual color system enables one to rapidly differentiate false positive beads from true positive beads, resulting in a much more efficient use of the microsequencer.
Subject(s)
Peptides/analysis , Recombinant Proteins/analysis , Amino Acid Sequence , Color , Immunologic Techniques/instrumentation , Molecular Sequence DataABSTRACT
When all L-amino acid (aa) random peptide libraries synthesized on solid-phase particles were screened (Selectide Technology), we identified several peptide ligands (YG_F_) that interacted specifically with an anti-beta-endorphin monoclonal antibody (clone 3E7) (single-letter aa symbols; symbols '_' indicate variable aa). Here, we report on the screening of three different D-aa-containing pentapeptide libraries (XxXxX, xXxXx and xxxxx, wherein X = L-aa, and x = D-aa) with the same antibody, in which several D-aa-containing ligands were identified. The binding affinities of many of these D-aa-containing ligands were as least two orders of magnitude lower than that of YGGFL, for which the Ki is 17.5 nM.