A HUMAN PANCREATIC SECRETORY TRYPSIN-INHIBITOR PRESENTING A HYPERVARIABLE HIGHLY CONSTRAINED EPITOPE VIA MONOVALENT PHAGEMID DISPLAY

Hypervariable gene banks displaying ligands which can be used for affinity optimisation are valuable resources for examining shape space. They have added value if the ligand is small, if there is extensive information on its tertiary structure and if the variable region is highly constrained. These features would be expected to stabilise complexes by reducing the dissociation constants and to facilitate their use as 'lead substances' for the development of synthetic mimetics. The synthesis and characterisation of such phagemid-display banks is described here, in which the variable region is a 7-amino acid (aa) (pSKAN8-HyB/C) or 8-aa (pSKAN8-HyA) extended peptide held between two disulfide bridges at the exposed tip of the human pancreatic secretory trypsin inhibitor (PSTI). A phagemid pSKAN8 was created which contains a fusion between the PSTI and M13 pIII protein-coding genes. Cassettes containing the sequences (NNK)(8) [HYA], (NNK)(7) [HyB] or (NNK)(6)GTT [Hy-C] (where K=G or T) were used to randomize the aa coding region in the trypsin-inhibitory loop (aa 17 to 23) of PSTI. Some 31 million individual clones were generated in a mutS Escherichia coli strain kept as frozen cell stocks. Analysis of controls which had not undergone selection showed very low levels of deletion. The quality of the hypervariable region and bias of codon usage was quantified by DNA sequencing. It was estimated from SDS-PAGE that hybrid protein was represented statistically at a frequency of one molecule per two phagemid particles. The functionality and reproducibility of the system was demonstrated by trypsin-binding of the original vector and in selecting novel chymotrypsin inhibitors from the banks.