Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 3.37

REPERTOIRES OF RECOMBINANT ANTIBODIES: A NOVEL APPROACH FOR FUNCTIONAL PROTEOMICS STUDIES APPLIED TO PLANT PHYSIOLOGY

DESIDERIO A., VILLANI M.E., LOPEZ M.

ENEA, CR Casaccia, UTS BIOTEC, Via Anguillarese 301, 00060 Santa Maria di Galeria (Roma)

recombinant antibodies, proteomics, phage display, molecular repertoires

The techniques of molecular genomics have been considerably developed in the last years offering a precious tool for the study of several biological systems, including plant physiology and physiopathology. Nevertheless, information deriving from DNA sequences gives only a static view of cell expression potential. Proteomics allows to correlate the enormous amount of genetic data with the relative expression products and to evaluate the interaction among them through the comparative analysis of cells in different physiological condition. This kind of studies requires an effective molecular device for rapid identification of proteins and for the study of their function.

In order to selectively identify a specific protein in complex mixtures of biological samples, molecular repertoires of recombinant antibodies can be used. Single chain Fv recombinat antibodies (scFv), characterised by small molecular dimensions and high binding capacity, demonstrated a great applicative potential. This recombinant antibody can also be expressed on the surface of bacteriophages as fusion product with the proteins of viral capside (‘phage display’). Phages expressing scFv with different binding specificities represent a molecular repertoire, from which it is possible to select antibodies through recursive cycles of selection, amplification and elution (‘biopanning’). The selected molecules can then be easily expressed in soluble form, identified and purified using appropriate tag peptides. With this technology specific binders can be fast obtained in virtually unlimited amount and used both for in vivo identification and localisation of proteins and for immunomodulation of specific protein function.

One of the main limit in antibody use as in vivo interfering molecules is represented by the difficulty to functionally express this naturally secreted protein in cell cytoplasm. In this reducing environment disulphide bonds cannot be formed and folding enzymes are absent, often leading to insoluble protein aggregates that are rapidly degradated by the cell. This represents a serious restriction for many applications requiring the interaction with target molecules expressed in cytosol compartment.

Recently a repertoire of recombinant antibodies has been constructed in ENEA, using the scaffold of a scFv antibody with remarkable molecular characteristics. This molecule, derived from a mouse monoclonal antibody, showed exceptional molecular properties, such as a functional expression in cytoplasm of bacterial, yeast and plant cells and a long in vivo half-life. Moreover denaturation/renaturation in vitro studies demonstrated a high intrinsic molecule stability. The framework of this antibody demonstrated to be an excellent scaffold for the construction of new antibodies suitable as intracellular immunomodulation (“intrabodies”) and/or diagnostics. By means protein engineering of defined position (only four residues in CDR3 of both VH and VL domain) of the hypervariable antibody regions, involved in antigen recognition, a molecular repertoire was obtained with new specificities and biochemical characteristics similar to the cognate antibody (high stability, solubility and functionality in the cytoplasm).

Antibodies directed against plant viruses have been already isolated from this repertoire and are currently being used in interference studies of infection development. In the same way other specific reagents can be obtained to investigate molecular processes involved in plant-pathogen interaction (i.e. resistance response, infection mechanism), in stress responses or in plant growth development (i.e. light response, metabolites accumulation, fruit maturation).