Geschäftsstelle des SPP 1623
Tel.: 030 94793 180
Fax: 030 94793 188
Robert-Rössle Str. 10, Geb. 81
The incorporation of unnatural designer amino acids into proteins at defined sites in living cells and organisms, via genetic code expansion, has provided new insights into biological processes that are challenging or impossible to address by classical approaches. Recent developments, such as genome engineering in Escherichia coli and knockout of bacterial release factor RF1 have seen tremendous progress in making amber suppression very efficient for incorporation of unnatural amino acids (UAAs) in bacterial hosts. The efficient, high protein yielding, site-specific incorporation of UAAs into proteins in mammalian cells, represents however still an outstanding challenge. A recent study reported increased protein levels, demonstrating that genetic code expansion can be optimized for mammalian cells.
So far, however, all systems for site-specifically incorporating UAAs into proteins in mammalian cells rely on transient transfections. This leads to highly variable expression yields between cells, limits its use to small-scale experiments, offers no (or only laborious) high content (e.g. proteomics) or screening possibilities and can thereby not be used to study dynamic processes (e.g. differentiation) in biological systems.
To overcome these limitations we propose to develop a stable, genomically integrated system for the site-specific introduction of UAAs into proteins in mammalian cells. We will furthermore develop new approaches and bioorthogonal chemistries for studying protein-protein and selectively protein-DNA interactions in live cells by introducing new photo-crosslinking moieties. Combining these efforts will allow us to study transient and weak protein interactions as well as the spatio-temporal localization and kinetics of target proteins during dynamic processes such as cellular differentiation.
To achieve this we aim to (i) integrate the Pyrrolysyl tRNA Synthetase (PylRS)/tRNA system stably into precise genomic loci of mouse and human cells by using a CRIPSR/Cas based genome engineering approach, (ii) develop new light induced bioorthogonal reactions that allow specific DNA-protein crosslinking via [2+2] photocycloaddition with high spatio-temporal resolution, and finally (iii) combine the stable transgenic PylRS/tRNA system with the newly developed derivatives for DNA specific photo-crosslinking to study the spatio-temporal localization, interactions and dynamics of the DNA-methyltransferase DNMT3B during epiblast differentiation. Our study will expand the utility of genetic code expansion for studies of highly dynamic processes as well as large-scale experiments in mammalian cells. The novel chemistries we aim to develop will be valuable tools for the study of localization and kinetics of DNA interacting factors.
Dr. Sebastian Bultmann
Biozentrum / Ludwig Maximilians University Munich
Tel.: +49 89 218 074 233
Fax: +49 89 218 074 236
Prof. Dr. Kathrin Lang
Technische Universität München / Fakultät für Chemie
Tel.: +49 89 289 13 836