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Projekt
Directed evolution of the S1 subsite specificity of trypsin
Antragsteller
Professor Dr. Frank Bordusa; Professor Dr. Milton T. Stubbs
Fachliche Zuordnung
Biochemie
Förderung
Förderung von 2006 bis 2011
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 30307660
Proteinases play a central role in many physiological, pathological and biotechnological processes. The family of trypsin-like serine proteinases (also known as SI A proteinases) has been the subject of extensive study, in particular with regard to proteolytic specificity. Most if not all members of this family characterised to date exhibit a pronounced preference for the substrate residue immediately preceding the peptide bond to be cleaved (the PI residue). Previous investigations by the applicants have concentrated on structure-based enzyme design to manipulate the properties of trypsin towards altered specificity or peptide ligation reactions; although fruitful, limitations are revealed, suggesting that a directed evolution approach is more suitable to the establishment of previously unknown specificities.As a prelude to the design of proteolytic enzymes with designed specificity, the current project sets out to establish PI specificities for L-Gln and D-Arg using trypsin as a scaffold; neither specificity exists for SI A family members to date. The former L-Gln trypsin will serve as a model system for the production of protein C variants capable of cleaving after Gin; replacement of a single Arg residue in factor V Leiden is responsible for hereditary thrombophilia in up to 20% of patients suffering from venous thromboembolism, and is a major risk factor for thrombosis. The latter D-Arg trypsin will form the basis for an expanded repertoire of ligands for substrate-mediated peptide and protein synthesis. A library of variants exhibiting changes in the primary specificity pocket will be constructed. Selection will take place initially through an activity and specificity screening of periplasmatic expressed trypsin species using an optimized E. coli expression system and Ac-Xaa-pNa substrates containing the desired PI residue (i.e. Xaa: L-Gln or D-Arg). In vitro selection using phage display will also be carried out using immobilised forms of the L-Gln- and D-Argcontaining BPTI variants, as BPTI binds to trypsin in a substrate-like manner. Variants that survive the binding tests will be subject to verification using the specifically synthesised chromogenic substrates. Subsequent crystallisation and X-ray structural analysis in complex with low molecular weight inhibitors and BPTI variants will guide further experiments to enhance binding and catalytic ability.
DFG-Verfahren
Schwerpunktprogramme
