Biol. Chem., Vol. 380, pp.101 –105, January 1999 · Copyright © by Walter de Gruyter · Berlin · New York Short Communication Phage Display Selection of P1 Mutants of BPTI Directed against Five Different Serine Proteinases Liliana Kiczak 1 , Katarzyna Koscielska 1 , Jacek Otlewski 1, *, Marcin Czerwinski 2 and Michal Dadlez 3 1 Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland 2 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, Wroclaw, Poland 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-126 Warszawa, Poland * Corresponding author The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mu- tants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the sur- face of M13 phage. The wild type of displayed inhibitor monovalently and strongly 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 ser- ine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pan- creatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azuro- cidin, the phage display selection allowed determina- tion of the P1 specificity of this protein with the follow- ing frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln. Key words: BPTI / Phage display / Protein inhibitors / Protein library / Serine proteinases. The phage display strategy enables the presentation of large peptide and protein libraries on the surface of phage particles, from which molecules with desired functional property(ies) can be rapidly selected (Barbas and Burton, 1993). The great advantage of this method is a direct link- age between an observed phenotype and an encapsulat- ed genotype, which allows for a rapid determination of selected sequences. The phage display approach has be- come a powerful tool in generating highly potent biomole- cules (Nedwidek and Hecht, 1997), including searching for specific antibodies (Nygren and Uhlén, 1997), and explor- ing protein – protein interactions (Allen et al., 1995). Protein inhibitors of serine proteinases that form cano- nical complexes with their target enzymes (Bode and Hu- ber, 1992) are an excellent system to apply combinatorial procedures offered by phage display. In particular, the family of Kunitz inhibitors homologous to BPTI has been already extensively studied using this approach. As a result, many amino acid sequences that ensure strong and/or specific binding to a number of serine proteinases have been found (Roberts et al., 1992; Dennis and La- zarus, 1994a and b). Interestingly, unexpected results of selection have also been observed for kallikrein (Markland et al., 1996), thrombin (Markland et al., 1996) and chymo- trypsin (Kossiakoff et al., 1993; Scheidig et al., 1997). In particular, high frequencies of the variants bearing Asn, His or Arg at P1 have been found for chymotrypsin (Kos- siakoff et al., 1993; Scheidig et al., 1997). These side chains are known to bind about 10 3 to 10 4 -fold weaker than optimal Tyr and Trp (Lu et al., 1997). Surprisingly, neither Tyr nor Trp have been observed at all after the selection. We are currently involved in applying site-directed mu- tagenesis and phage display methods to select BPTI vari- ants against several serine proteinases and to determine respective association energies. As the P1 side chain pro- vides up to 40 – 70% of the total association energy to the complex formation (Lu et al., 1997; Qasim et al., 1997), we decided first to analyze a small library of P1 mutants of BPTI to determine whether thermodynamically optimal side chains can be selected. The enrichments were per- formed on four serine proteinases (bovine trypsin and chymotrypsin, porcine pancreatic and human leukocyte elastases) for which high quality thermodynamic data on binding to inhibitors with 20 different amino acids at P1 are already available (Lu et al., 1997). We also performed the screening on human azurocidin, a serine proteinase-like protein of unexplored specificity, which during evolution lost the catalytic triad (Ser195Gly, His57Ser). The pComb3H phagemid system was obtained from the Scripps Research Institute, La Jolla, CA (Barbas and Burton, 1993). This vector was originally constructed for expression of antibody Fab fragments on the surface of M13 phage and contained ColE1-ori, f1-ori, amp r , and the lac promotor for controlling expression of fusion genes: ompA – light chain and pelB – heavy chain – pIII 230-406 . To obtain the BPTI presentation system we substituted the heavy chain gene of pComb3H with the PCR-amplified