title: Trypsin mutants for structure-based drug design: expression, refolding and crystallisation.
abstract: New techniques in drug discovery are essential for the fast and efficient development of novel innovative drugs to deal with the challenges of the future. Structure determinations of various members of serine proteinases have provided a basis for computer-based drug design within this class of enzymes. In many proteins of interest, however, this course is blocked through a lack of suitable crystals. As a strategy for circumventing such problems, we have investigated the use of surrogate proteins for studying protein-ligand interactions. To test the feasibility of this approach, we have chosen bovine trypsin as a scaffold to reconstruct the ligand binding site of factor Xa. The simple modular design of trypsin, its readiness to crystallise and straightforward handling lends itself to such drug design by proxy. The expression, folding, purification, crystallographic and kinetic characterisation of bovine trypsin forms with factor Xa phenotype are presented.
authors.affiliation: Institut für Pharmazeutische Chemie der Philipps-Universität Marburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rauh
authors.forename: Sabine
authors.initials: S
authors.lastname: Reyda
authors.forename: Gerhard
authors.initials: G
authors.lastname: Klebe
authors.forename: Milton T
authors.initials: MT
authors.lastname: Stubbs
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003460:Crystallization; D015195:Drug Design; D015951:Factor Xa / Q000737:chemistry; D065427:Factor Xa Inhibitors; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D020673:Protein Renaturation; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D065427:Factor Xa Inhibitors; D014357:Trypsin; D015951:Factor Xa