Berger Group

An intense focus of current biological research efforts is the elucidation of protein interaction networks (interactome). Many large multiprotein complexes are discovered. This poses considerable challenges for molecular level studies, in particular for eukaryotic multiprotein complexes with intracellular quantities refractory to large-scale extraction from source. Our research is focused on developing new technologies to obtain, within a reasonable time-frame, well-defined and homogeneous samples of human multiprotein assemblies in transcription and hereditary disease, which we then use for high-resolution structural and functional analyses.

Our major methodologies include molecular biology and X-ray crystallography. However, we readily apply techniques from other fields that are required for our research, both by ourselves and through collaborative efforts. A prerequisite for successful structural study of many complexes, both by electron microscopy and X-ray crystallography, is production of homogeneous, stable specimens. Present recombinant expression methods often require considerable investment in both labour and materials before multiprotein expression, and after expression and biochemical analysis do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have introduced MultiBac, a modular, baculovirus-based system specifically designed for eukaryotic multiprotein expression (Berger et al., Nat. Biotechnol. 22, 2004). Recently, we have harnessed homologous and site-specific recombination methods in tandem for all steps involved in multigene assembly, thus providing a flexible, automatable platform for generation of multiprotein expression vectors and their rapid regeneration for revised expression studies. We have successfully implemented all steps involved in a robotics setup. By using our technology, we produced numerous large multiprotein assemblies in sufficient quantity and quality for structural studies, including the presumed ~700 kDa heterodecamer scaffold of human TFIID general transcription factor.

Future projects and goals

At EMBL Grenoble, we will continue to advance our expression technologies to entirely automate and standardise the process of production for eukaryotic gene regulatory multiprotein complexes including the entire human TFIID holoenzyme and its various isoforms. In collaboration with the Schaffitzel group, we will subject the complex specimens produced to electron microscopic analyses. We will use homogeneous complexes thus identified for X-ray crystallography.

By enlisting state-of-the-art mass spectrometric methods from systems biology, we will address a further bottleneck in complex crystallography, namely the challenge of defining crystallisable core assemblies of multiprotein complexes in a reasonable time frame (in collaboration with ETH Zürich). Also, we will expand our multiprotein expression strategies to prokaryotic and mammalian hosts.