Figure: We develop and utilise advanced, automated technologies to produce eukaryotic multiprotein complexes for structural and functional analysis by a variety of methods including X-ray crystallography.
The Berger group studies eukaryotic multiprotein assemblies in transcription regulation, develops technologies to produce them recombinantly and subjects them to high-resolution structural and functional analyses.
Previous and current research
Human gene transcription requires the controlled step-wise assembly of the pre-initiation complex (PIC), comprising a large ensemble of proteins and protein complexes including RNA Pol II and the general transcription factors. TFIID is the first general transcription factor to bind to gene promoters, and is a cornerstone of PIC assembly. However important, we lack detailed knowledge of its molecular architecture and interactions with cellular factors. Endogenous TFIID is scarce and heterogeneous. Therefore, we created new technologies to produce TFIID and similar multiprotein complexes recombinantly. Notably, our MultiBac system – a modular, baculovirus-based technology specifically designed for eukaryotic multi-protein expression – is now used in many labs worldwide. Recently, we determined the architecture of the 700 kDa heterodecameric human TFIID core complex by combining MultiBac-based production with cryo-EM, X-ray crystal analysis, homology modelling, and proteomics data.
We collaborate with groups from academia and industry to further automate labour-intensive steps in the multiprotein complex structure determination process, and have harnessed homologous and site-specific recombination methods for assembling multigene expression plasmids. We have developed ACEMBL, a proprietary automated suite for multigene recombination on our TECAN EvoII platform. It allowed us to produce numerous large multiprotein assemblies for structural studies, and to expand our multiprotein expression strategies to prokaryotic and mammalian hosts.
Future projects and goals
We work towards entirely automating and miniaturising the production-process for eukaryotic multiprotein complexes including the entire human TFIID holocomplex, its various isoforms and other components of the preinitiation complex. In collaboration with the Schaffitzel Team and the Schultz Group (IGBMC Strasbourg), we subject the complex specimens produced to electron microscopic and X-ray crystallography analyses to understand their physiological function, and further our findings by in vitro and in vivo biochemical analysis.
Using state-of-the-art mass spectrometric methods we are developing MultiTRAQ, a new technology to address the challenge of defining crystallisable core assemblies of multiprotein complexes in a reasonable time frame. Another recent project line in our lab exploits synthetic biology techniques for genome engineering, with the aim of creating disruptive platforms for recombinant protein production, for both academic and industrial applications.
Recently, we implemented ComplexLink, our polyprotein-based technology to enable production of hitherto inaccessible protein complexes. We are applying ComplexLink to high-value targets such as viral polymerases and multisubunit kinases.