title: Affinity purification of fibrinogen using a ligand from a peptide library.
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.
authors.affiliation: Department of Chemical Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Partner's Building I, Suite 3200, Box 7006; Raleigh, North California 27695-7006, USA.
authors.forename: Deborah B
authors.initials: DB
authors.lastname: Kaufman
authors.forename: Marc E
authors.initials: ME
authors.lastname: Hentsch
authors.forename: George A
authors.initials: GA
authors.lastname: Baumbach
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Buettner
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Dadd
authors.forename: Ping Y
authors.initials: PY
authors.lastname: Huang
authors.forename: David J
authors.initials: DJ
authors.lastname: Hammond
authors.forename: Ruben G
authors.initials: RG
authors.lastname: Carbonell
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D005340:Fibrinogen / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D012492:Salts; D013696:Temperature
chemical_list: D019151:Peptide Library; D010455:Peptides; D012492:Salts; D005340:Fibrinogen