title: Affinity purification of tissue plasminogen activator using transition-state analogues.
abstract: The search for a simple affinity ligand to purify tissue plasminogen activator (tPA) was facilitated by a solid-phase synthesis approach. A large variety of tripeptide ligands containing argininal were synthesized on agarose gels containing a spacer with carboxy terminal. The immobilized ligands were easy to test with urokinase, and tPA. While a number of sequence combinations showed initial binding by tPA, only a few resulted in tight binding corresponding to a hemiacetal linkage with the active site serine. Hydrophobic residues, especially aromatics, flanking the N-side of argininal gave rise to ligands which were bound strongly by tPA. A gel containing D-Phe-D-Phe-Argal (an aldehyde derivative of arginine) was very effective in purifying tPA derived from cell culture media at small scale (milligrams) and at large (multi-grams).
authors.affiliation: Protein Biochemistry Department, SmithKline Beecham Pharmaceuticals R&D, King of Prussia, PA 19406-0939.
authors.forename: A
authors.initials: A
authors.lastname: Patel
authors.forename: M
authors.initials: M
authors.lastname: O'Hara
authors.forename: J E
authors.initials: JE
authors.lastname: Callaway
authors.forename: D
authors.initials: D
authors.lastname: Greene
authors.forename: J
authors.initials: J
authors.lastname: Martin
authors.forename: A H
authors.initials: AH
authors.lastname: Nishikawa
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D006801:Humans; D007202:Indicators and Reagents; D008024:Ligands; D008969:Molecular Sequence Data; D013917:Thrombin; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator
chemical_list: D005782:Gels; D007202:Indicators and Reagents; D008024:Ligands; D014357:Trypsin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator