Protein Targeting Compounds

Marschall, Andrea L J, Dübel, Stefan, Böldicke, Thomas, Andrea L. J. Marschall, Stefan Dübel, Thomas Böldicke, Marschall, Andrea L. J.

ER intrabodies are recombinant antibody fragments produced and retained in the endoplasmatic reticulum (ER) of a cell or an organism with the purpose to induce phenotypes generated by interfering with the intracellular processing or by changing the location of the recognized antigen. The most common application is the generation of functional knockdowns of membrane proteins, which cannot reach their natural location on the cell surface when they are retained in the ER by the intrabody. Phenotypes generated by interfering with the secretion of extracellular or plasma proteins can be analyzed in a similar way. So far, most ER intrabody studies relied on scFv fragments subcloned from hybridoma lines. Recently, several large international research consortia have started to provide antibodies, with the final goal to cover substantial parts of the human proteome. For practical reasons of throughput and effort, in these consortia the most appropriate method to generate the necessary large numbers of monoclonal antibodies is in vitro selection, typically employing phage or yeast display. These methods provide the antibody genes right from the start, thereby facilitating the application of ER antibody approaches. On the other end, the first transgenic mice expressing an ER intrabody has recently been described. This moves the ER intrabody approach finally to level with classic in vivo knockout strategies - but also offers novel capabilities to the researchers. Promising new perspectives may originate from the fact that the knockdown is restricted to the protein level, that a graded knockdown strength can be achieved, or that the targeting of individual posttranslational modifications will be possible with previously impossible specificity. Finally, the link of today's high throughput recombinant antibody generation to a knock down phenotype is now possible with a single cloning step. It can therefore be expected that we will see a much quicker growth of the number of successful applications of ER intrabody technology in the near future than it has been seen in its first two decades.