1.1 - The Aim of the Research. Antibodies can be ectopically expressed as secreted proteins in cells that do not normally express them to interfere with extracellular antigens and/or as intracellular proteins targeted to different intracellular compartments to inhibit intracellular gene products: this has lead to an effective protein knock-out technology. Expression of antibodies inside cells has been used successfully to ablate protein function. The performance of antibodies that are intracellularly expressed is however somewhat unpredictable because the reducing environment of the cell cytoplasm in which they are forced to work prevents some antibodies, but not others, to fold properly. For this reason, a selection procedure for the isolation of antibodies able to fold correctly and to bind antigens under condition of intracellular expression would be highly desirable. The development of this novel selection procedure could exploit methods to monitor intracellular protein – protein interactions, for instance the yeast two-hybrid technology. Such a system would greatly facilitate the isolation of candidate antibodies for intracellular antibody down-stream applications in the system of interest, and would lend itself to the development of cell-based high-throughput screening procedures in functional genomics applications. In view of this, the creation of a new technology that allows the isolation of intracellular scFv fragments, based on their ability to bind antigen under conditions of intracellular expression has become an important goal both for functional genomics and for gene therapy, to fulfill the requirement of antibodies with improved thermodynamic stability and solubility properties. 1.2 - Results The following chapters will describe all the steps taken in order to achieve the final goal: the development of the intrabody trap technology (ITT), an in vivo selection and assay procedure for functional intracellular antibodies using a two-hybrid approach. In the first part of the project we find that several characterized antibodies can bind their target antigen in eukaryotic cells when expressed in the two-hybrid format and we have been able to isolate intracellular binders from panels of scFv, all of which can bind antigen in vitro. Furthermore, we showed a model selection in which a single chain scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate isolation of intrabody specificity from complex mixtures. The results form the basis of the “intrabody trap technology”, whereby many different, specific and functional antibodies can be isolated in vivo under condition of intracellular expression. The second part of the thesis reports the demonstration that ITT can be effectively applied to the de novo selection of functional intrabodies, by performing a real selection from polyclonal scFv fragments partially enriched from a large naive phage library by antigen panning. The experimental procedure described in this thesis has allowed the selection of intrabodies against a protein antigen involved in Alzheimer’s disease (AD), the microtubule associated protein tau, the main components of the paired helical filaments (PHFs), found in neurofibrillary lesions in AD brains. In particular the aim of the work was to isolate scFv fragments against a tau fragment, which displays apoptotic capacity in different cellular context.
|Titolo:||The Development of Intrabody Trap Technology (ITT) for Functional Genomics|
|Data di pubblicazione:||24-ott-2000|
|Appare nelle tipologie:||8.1 PhD thesis|