title: Design and study of peptide-ligand affinity chromatography adsorbents: application to the case of trypsin purification from bovine pancreas.
abstract: The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 micromol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 micromol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 microM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 microM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 micromol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.
authors.affiliation: Enzyme Technology Laboratory, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece.
authors.forename: T
authors.initials: T
authors.lastname: Makriyannis
authors.forename: Y D
authors.initials: YD
authors.lastname: Clonis