© 2005 The Japanese Biochemical Society
Regular Paper |
Complete Substitutional Analysis of a Sunflower Trypsin Inhibitor with Different Serine Proteases
1 Department of Biochemistry, Medical Faculty Charité, Humboldt University of Berlin, Monbijoustr. 2, 10117 Berlin, Germany; and 2 Department of Medical Immunology, Medical Faculty Charité, Humboldt University of Berlin, Schumannstr. 20-21, 10117 Berlin, Germany
* To whom correspondence should be addressed. Tel: +49-30-450528153, Fax: +49-30-450528909, E-mail: wolfgang.hoehne{at}charite.de
Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis. Inhibitor variants optimized for elastase or proteinase K inhibition by several rounds of substitutional analyses exhibit Ki values in the micromolar range and high specificity for the corresponding protease. The results of this easy-to-perform assay can be used to design an improved peptide library using classical methods.