No abstract

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

No abstract

We have developed a new resin for peptide synthesis that can be used to synthesize and evaluate directly combinatorial peptide libraries for binding target proteins. Fidelity of the peptide synthesis using this hydrophilic resin is comparable to polystyrene‐based resins. Peptide libraries synthesized on this resin were probed by a two color PEptide Library Immunostaining Chromatographic ANalysis (PELICAN) technique for sequences binding the serine protease Factor IX zynogen. This PELICAN technique readily distinguishes between beads interacting with the reagents for target detection (blue beads) from those beads specific for the target protein itself (red beads). Validation of the PELICAN technique, as well as purification of Factor IX from plasma, is demonstrated utilizing this resin. ©Munksgaard 1996.