Phage display selection of P1 mutants of BPTI directed against five different serine proteinases

The P1 position of protein inhibitors and oligopeptide substrates determine s, to a large extent, association energy with many serine proteinases. To t est the agreement of phage display selection with the existing thermodynami c data, a small library of all 20 P1 mutants of basic pancreatic trypsin in hibitor (BPTI) was created, fused to protein III, and displayed on the surf ace of M13 phage. The wild type of displayed inhibitor monovalently and str ongly inhibited trypsin with an association constant of K-a = 3 . 10(11) M- 1. The library was applied to select BPTI variants active against five seri ne proteinases of different specificity (bovine trypsin and chymotrypsin, h uman leukocyte and porcine pancreatic elastases, human azurocidin), The res ults of enrichment with four proteinases agreed well with the available the rmodynamic data. In the case of azurocidin, the phage display selection all owed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln.