Establishing platforms for the production of recalcitrant proteins for functional and structural studies

Background.

Proteins are composed of amino acid residues and responsible for a majority of important cellular functions. To fully understand how proteins carry out these diverse processes, it is essential to combine functional studies with an investigation of their three-dimensional structure. As many proteins play critical roles in the development of disease, an understanding of the protein structure at an atomic level not only provides increased understanding of cellular functions but can lead to further understanding of pathological mechanisms and be critical for the development of therapeutic drugs. Additionally, more and more enzymes are used in daily life, for example in wastewater-treatment or food production

Aims and methods.

The production of proteins is presently performed by recombinant molecular biology techniques. However, every protein behaves differently and as functional and structural studies of proteins and their complexes require milligram amounts of active, chemically and conformationally pure protein, the production of the proteins is still a major bottleneck. The second bottleneck is the generation of diffraction quality crystals for structural biology studies employing macromolecular crystallography.

To tackle these challenging bottlenecks we will establish a platform for production of soluble and stable proteins and/or protein complexes based on parallel screening of nested constructs in E. coli and employing the cell-free expression systems. Furthermore, we will develop and establish a customized crystallization screening based on the biophysical properties of individual targets.

The efficiency of our protein production and crystallization platform will be assessed and subsequently used on a on a series of different kinds of proteins, mainly building the cytoskeleton of cells, antibodies membrane transporters and enzymes involved in protection from reactive oxidative species.