Reconstitution of tyrosine kinase signaling in cell-free systems: Synthetic membrane protein dimerization and lipid modification

The Main Goals of the project are:

  • Reconstitution of EGFR depended signaling pathways in cell-free systems
  • In vitro generation and chemical modification of constitutively active EGF-Receptor (via genetically encoded unnatural amino acids utilized to dimerize the receptor permanently) and its downstream effector RhoA (via the introduction of lipid moieties facilitated by unnatural amino acids)
  •  Reconstitution of integral membrane proteins and membrane associated proteins in lipid bilayers by combination of chemical methods and naturally occurring posttranslational modifications in cell-free systems (via genetically encoded unnatural amino acids and novel heterobisfunctional linker chemistries)
  • Development of novel heterobisfunctional linker chemistries for protein dimerization- Implementation of novel tRNA/tRNA synthetase pairs for the incorporation of unnatural amino acids into proteins in various in vitro translation systems
  • Monitoring functionality of in vitro synthesized and constitutive active membrane proteins through detection of phosphorylation (Antibody based Assays) and Protein-Protein interaction analysis (mass spectrometry Assays)- Identification of novel inhibitory components with regard to the EGF-receptor’s downstream signaling pathway.

Dr. Stefan Kubick
Fraunhofer Institute for Cell Therapy and Immunology (IZI)
Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam
Department of Cell-free Bioproduction

Tel.: +49 331 58187 - 306
Fax: +49 331 58187 - 399

Email Dr. Kubick

Dr. Stefan Schiller
Freiburg Institute for Advanced Studies
Albert-Ludwigs-Universität Freiburg

Tel.: +49 761 203 97405
Fax: +49 761 203 97451

Email Dr. Schiller

Publications within the SPP 1623 project

A. Zemella, T. Richter, L. Thoring, S. Kubick
Protocol 2019, DOI: 10.1007/978-1-4939-9121-1_4
A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties
Link to the article

A. Zemella, L. Thoring, C. Hoffmeister, M. Samalikova, P. Ehren. D.A. Wüstenhagen, S. Kubick
Scientific Reports 2018, 8, Art. no. 8514
Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin
Link to the article

A. Zemella, S. Grossmann, R. Sachse, A. Sonnabend, M. Schaefer, S. Kubick
Scientific Reports 2017, 7:3740, DOI: 10.1038/s41598-017-03955-8
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses
Link to the article

L. Thoring, D.A. Wüstenhagen, M. Borowiak, M. Stech, A. Sonnabend, S. Kubick
PLoS ONE 2016, 11(9), e0163670
Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.
Link to the article

R.B. Quast, B. Ballion, M. Stech, A. Sonnabend, B.R. Varga, D.A. Wüstenhagen, P. Kele, S.M. Schiller & S. Kubick
Sci. Rep. 2016, 6, 34048
Cell-Free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.
Link to the article

R.B. Quast, A. Sonnabend, M. Stech. D.A. Wüstenhagen, S. Kubick
Sci. Rep. 2016, 6, 30399
High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format.
Link to the article

R.B. Quast, D. Mrusek, C. Hoffmeister, A. Sonnabend, S. Kubick
FEBS letters 2015, 589 (15), 1703-1723
Cotranslational incorporation of non-standard amino acids using
cell-free protein synthesis
Link to the article

A. Schreiber, M.C. Huber, H. Cölfen, S.M. Schiller
Nature Comm. 2015, DOI: 10.1038/ncomms7705
Molecular protein adaptor with genetically encoded interaction-sites guiding the hierarchical assembly of plasmonically active nanoarchitectures
Link to the article

R.B. Quast, O. Kortt, J. Henkel, S.K. Dondapati, D.A. Wüstenhagen, M. Stech, S. Kubick
J. of Biotech. 2015, 203, 45-53
Automated production of functional membrane proteins using eukaryotic cell-free translation systems
Link to the article

M.C. Huber, A. Schreiber, P. von Olshausen, B.R. Varga, O. Kretz, B. Joch, S. Barnert, R. Schubert, S. Eimer, P. Kele, S.M. Schiller
Nature Chemicals 2015, 14(1), 125,132
Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments
Link to the article

M. Stech, S. Kubick
Antibodies 2015, 4, 12-33
Cell-Free Synthesis Meets Antibody Production: A Review
Link to the article

M. Huber, A. Schreiber, K. Benz, S.M. Schiller
Biomat. 2014, 35,31, 8767-8779
Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid materials
Link to the article

S.F. Frenz, R. Sachse, T. Schmidt, S. Kubick
Biochimica et Biophysica Acta2014, 1838, 1382-1388
Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes
Link to the article

R.B. Quast, I. Clausnitzer, H. Merk, S. Kubick, M. Gerrits
Analytical Biochemistry2014, 451, 4-9
Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems
Link zum Artikel

S.K. Dondapati, M. Kreir, R.B. Quast, D.A. Wüstenhagen, A. Brüggemann, N. Fertig, S. Kubick
Biosens Bioelectron2014, 59, 174-183
Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system.
Link zum Artikel

S. Bechlars, D.A. Wüstenhagen, K. Drägert, R. Dieckmann, E. Strauch, S. Kubick
Toxicon 2013, 76, 132-142
Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus
Link zum Artikel

M. Stech, H. Merk, J.A. Schenk, W.F.M. Stöcklein, D.A. Wüstenhagen, B. Micheel, C. Duschl, F.F. Bier, S. Kubick
J. of Biotechnology 2012, 164, 220-231
Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system
Link zum Artikel

D.E. Zampatis, C. Rutz, J. Furkert, A. Schmidt, D. Wüstenhagen, S. Kubick, N.E. Tsopanoglou, R. Schülein
FEBS Letters2012, 586, 2351-2359
The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression
Link zum Artikel