Biomedical targets

Much of the drive of today's research in biology is directed towards a better understanding and treatment of human diseases. The Human Genome Project has produced an enormous body of information about the sequence of the human genome and in addition genome sequences of a large number of human pathogens, from tuberculosis to malaria, are also known. However, detailed information on the structure, function and interaction of the tens of thousands of proteins encoded by these genomes is required in order to fully exploit this new panoply of data for novel human disease treatments.

In the early stages of structural genomics, challenging targets such as those that are poorly soluble, unstable, intrinsically (partially) unstructured or are highly flexible are put aside because they do not crystallize. One way out is to purify and crystallize these proteins in complex with other proteins that reduce flexibility or improve the solubility of these 'higher hanging fruits'. Knowledge of the three-dimensional structures of key human or human pathogen proteins, is vital for speeding up the difficult task of discovering new therapeutics including antibiotics or anti-cancer drugs.

Chaperon (social): an adult who supervises one or more unmarried men or women during social occasions
Chaperon (headgear): a form of hood or versatile hat worn in Western Europe in the Middle Ages
Chaperon (protein): a protein that assists the non-covalent folding/unfolding in molecular biology
Chaperon (crystallography): a protein that assists another protein to crystallize

The occurrence of bona fide antibodies devoid of light chains in Camelidae was one of the major discoveries within our department. These so-called heavy-chain antibodies bind antigens solely with one single variable domain, referred to as VHH domain or Nanobody (Nb). Methods were developed to clone the VHH repertoire of an immunised dromedary (or llama) in phage display vectors, and to select the antigen-specific VHHs from these 'immune' VHH libraries. Recombinant Nanobodies are small (15kDa) and strictly monomeric. They bind the target with nM affinity, are stable and easy to manipulate. Moreover, the Nanobodies often bind to epitopes that are less immunogenic for conventional antibodies, such as the active sites of enzymes. Due to their small size, they also target areas that are not accessible to standard antibodies. Another advantage is that they generally bind conformational epitopes and that they are well expressed in bacterial expression systems so that they are cheaper and easier to produce in all kind of formats than standard monoclonal antibodies.

In the last years, we proved the principle that Nanobodies can be used as crystallization chaperones in the crystallization and structure determination of challenging target proteins that would prove unsolvable using more conventional strategies including proteins from larger molecular complexes, aggregating proteins, oligomerizing proteins and membrane proteins.

• Aggregating proteins: We use nanobodies as tools in research on amyloidogenic proteins. Work of the last years, especially on Human Lysosyme has shown that nanobodies are indeed very useful to study mechanisms of stabilization/destabilization and folding dynamics of (mutant) proteins exhibiting amyloidogenic behaviour.
• Oligomerizing proteins: For a number of intrinsically multimerizing proteins, we are currently screening nanobodies that stabilize the monomeric protomer. Such nanobodies are then used to solve the structure of the monomer.
• Membrane proteins: Any X-ray structure analysis of membrane proteins has to overcome two main obstacles, namely to provide sufficient amounts of the protein and to obtain well-ordered three-dimensional crystals. The coming years, we want to exploit the unique properties of Nbs to facilitate the production, the purification, the crystallization and the functional characterization of a set of GPCRs and MDRs.