Ulrich Reineke

Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

The antigen binding domain of non-idiotypic human anti-F(ab′)2 autoantibodies: study of their interaction with IgG hinge region epitopes

In previous studies we described a natural human IgG-anti-F(ab)2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab)2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab)2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.

Identification of miniproteins using cellulose-bound duotope scans

Protein domains or small molecules that mimic protein-protein contact sites are playing an increasing role in drug discovery, diagnostics and biotechnology. The identification of such protein mimics is based on two different strategies: (1) biologically as well as chemically prepared combinatorial libraries that are utilized for the de novo generation of novel sequences; and (2) approaches that are based on three-dimensional structures of endogenous protein ligands. Reduction of their size by truncation or deletion of amino acids which are not involved in the interaction and subsequent optimization by site-directed mutagenesis may also involve and library techniques. In contrast, this study describes an approach based on the primary structure of a protein ligand. One important technique for the mapping of protein-protein contact sites is the use of scans of overlapping peptides derived from a protein sequence (peptide scan, Fig. 1 left) which are subsequently tested for binding of the respective interaction partner [1]. This technique is the method of choice for the identification of linear binding sites where most of the important residues for the interaction are located within one stretch of the primary structure. In contrast, the key interacting residues of discontinuous binding sites are distributed over two or more binding regions which are separated in the protein sequence and only form the composite high affinty epitope upon protein folding. The mapping of discontinuous binding sites with peptide scans is very difficult, if not impossible, since peptides comprising single binding regions characteristically have very low affinities for the binding partner. Hence, we introduced a novel type of peptide library, called a duotope scan. The rationale of this scan is that a discontinuous binding site can only be mimicked adequately if two or more binding regions are connected in one molecule by a linker moiety resembling their spacing in the three-dimensional structure. Therefore, all possible combinations of two overlapping peptides from a conventional peptide scan are synthesized as one linear molecule i. E. combinatorial chemistry with peptides as second level building blocks (Fig. 1 right). The duotope scans are synthesized on continuous cellulose membranes by SPOT-synthesis [2], a highly parallel positionally addressable synthesis technique.