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)6GTT [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.

Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems.

UNLABELLED: A monoclonal antibody directed against the gene 3 product (pIII) of filamentous phage M13 was produced to study pIII-fusion protein expression in E. coli and its incorporation in the phage capsid. The protein was gel-purified from E. coli expression cultures harboring the genetic information of pIII under the control of an inducible lac promoter. To study pIII-fusion protein expression, phage display systems were applied in which either the whole pIII or the C-terminal half was used (McCafferty et al. (1990) Nature (London) 348, 552-554; Szardenings and Collins (1990) Gene 94, 1-7; Barbas and Lerner (1991) In: METHODS: Companion to METHODS in Enzymology, Combinatorial Immunoglobulin Libraries on the Surface of Phage (Phabs): Rapid Selection of Antigen-Specific Fabs, Vol. 2, Academic Press, Orlando, pp. 119-124). In all cases, the monoclonal antibody was able to detect the native and the recombinant protein in E. coli and on the phage tip using non-denaturing (ELISA) and denaturing (SDS-PAGE, immunoblot analysis) conditions. All selected pIII-specific monoclonal antibodies were found to be directed against epitopes within amino acids 198 to 406 of pIII, which is necessary for capsid incorporation and therefore included in all pIII-mediated phage display designs.

[Pain, a human and a medical problem (author's transl)]. / 1. Der Schmerz--ein menschliches und ärztliches Problem

Physiological, psychological and also psychiatric aspects of pain are of great importance for the surgical treatment of pain. The inhibition of pain conduction of physical and psychic stimuli makes it difficult to evaluate therapeutic procedures and forces us to apply adequate treatment in each single case.