Affinity purification of tissue plasminogen activator using transition-state analogues.
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).
Patel
A
A
Protein Biochemistry Department, SmithKline Beecham Pharmaceuticals R&D, King of Prussia, PA 19406-0939.
O'Hara
M
M
Callaway
J E
JE
Greene
D
D
Martin
J
J
Nishikawa
A H
AH
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
D005782:Gels; D007202:Indicators and Reagents; D008024:Ligands; D014357:Trypsin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator