The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands.
Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.
Lathrop
Julia Tait
JT
Jerome Holland Laboratory, American Red Cross Biomedical R&D, Rockville, MD 20855, USA. lathropj@usa.redcross.org
Fijalkowska
Iwona
I
Hammond
David
D
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D005167:Factor VII / Q000737:chemistry; D005189:False Positive Reactions; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
D001798:Blood Proteins; D008024:Ligands; D011506:Proteins; D005167:Factor VII