Hydrophobic sequences can substitute for the wild-type N-terminal sequenceof cystatin A (stefin A) in tight binding to cysteine proteinases - Selection of high-affinity N-terminal region variants by phage display

phage-display library of the cysteine-proteinase inhibitor, cystatin A, was constructed in which variants with the four N-terminal amino acids randoml y mutated were expressed on the surface of filamenteous phage. Screening of this library for binding to papain gave predominantly variants with a glyc ine residue in position 4. This finding is in agreement with previous concl usions that glycine in this position is essential for tight binding of cyst atin A to cysteine proteinases by allowing optimal interaction of the N-ter minal region of the inhibitor with the enzyme. In contrast. the first three residues of the variants obtained by the screening were more variable. Two variants were identified with similar affinities for papain as the wild-ty pe inhibitor, but with these residues, Val-Phe-Thr- or Ile-Leu-Leu. differi ng appreciably from those of the wild-type, Met-Ile-Pro. Other sequences of the N-terminal region, presumably mainly hydrophobic, can thus substitute for the wild-type sequence and contribute similar energy to the inhibitor-p roteinase interaction. The two variants binding tightly to papain differed in their affinity for cathepsin B, demonstrating that cystatin variants wit h increased selectivity for a particular target cysteine proteinase can be obtained by phage-display technology.