Biomedical subjects

S Panni

Publications and source records attributed to S Panni.

4 recordsLinked to original sources

PubMed2002

Mapping the C terminal epitope of the Alzheimer's disease specific antibody MN423.

The mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. However, until very recently, phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. Here we describe the use of two different techniques to analyze the known C terminal epitope specificity of MN423, a monoclonal antibody specifically staining truncated tau in Alzheimer's brain. Using a lambda phage based C-terminal random peptide library, and an intracellular expression library based on truncated tau, we show that this antibody has an absolute requirement for a glycine at position -3 with respect to the C terminus. Both methods give similar results, and identify other important residues in the binding site. However, affinity analysis of synthetic peptides revealed that the affinity of the antibody for identified tripeptides was far lower than the pentapeptide sequence in the native target, and that this in turn was considerably below the affinity for the native target itself. This suggests that molecular diversity methods may define minimum, but not necessarily complete epitopes. The methods described here have a general application to the analysis of antibody epitopes suspected to be found at the C terminus.

Aged↗

PubMed2001

Alternative bacteriophage display systems.

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm. Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis. This review describes and compares the properties of these alternative display systems.

Bacteriophages↗

PubMed2000

Domain repertoires as a tool to derive protein recognition rules.

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.

Amino Acid Motifs↗

PubMed1999

A novel peptide-SH3 interaction.

SH3 domains constitute a family of protein-protein interaction modules that bind to peptides displaying an X-proline-X-X-proline (XPXXP) consensus. We report that the SH3 domain of Eps8, a substrate of receptor and non-receptor tyrosine kinases, displays a novel and unique binding preference. By a combination of approaches including (i) screening of phage-displayed random peptide libraries, (ii) mapping of the binding regions on three physiological interactors of Eps8, (iii) alanine scanning of binding peptides and (iv) in vitro cross-linking, we demonstrate that a proline-X-X-aspartate-tyrosine (PXXDY) consensus is indispensable for binding to the SH3 domain of Eps8. Screening of the Expressed Sequence Tags database allowed the identification of three Eps8-related genes, whose SH3s also display unusual binding preferences and constitute a phylogenetically distinct subfamily within the SH3 family. Thus, Eps8 identifies a novel family of SH3-containing proteins that do not bind to canonical XPXXP-containing peptides, and that establish distinct interactions in the signaling network.

Adaptor Proteins, Signal Transducing↗