Cell-Free Protein Synthesis

Research in the field of cell-free protein synthesis deals with the synthesis of recombinant proteins in various cell-free systems. In order to analyze proteins, these must first be made available in a functionally active form. The expression of proteins in living cells, i.e. in vivo, is widely used, but does not always yield the desired results, since not every protein can be satisfactorily synthesized in cell cultures. Cell-free protein synthesis thus offers an efficient alternative to the expression of proteins in living cells, whereby the substances found within the cell are used to produce a certain target protein quickly and cost-effectively. The use of eukaryotic cell lysates has the special advantage of allowing the synthesis of proteins with post-translational modifications (PTMs). Particular emphasis is placed on the characterization, modification and functional examination of the cell-free produced antibody formats and membrane proteins. Cell-free systems are used in time-saving, highly parallelized methods to synthesize antibodies and membrane proteins and to immediately integrate these into functional analysis.

Protein Labeling & Modification

Antibody Technologies

Membrane Protein Synthesis

  • Production, optimization and cloning of »ready-to-express« DNA templates for cell-free protein synthesis
  • RNA synthesis (transcription, analysis and purification of mRNA)
  • Cell-free synthesis and characterization of recombinant antibody formats
  • Synthesis of recombinant antibody formats based on linear or circular DNA templates in eukaryotic in-vitro transcription/translation systems.
  • Parallel production of antibody formats in various reaction routes, e.g. in batch and dialysis mode or coupled (transcription and translation in one reaction system) and decoupled (transcription and translation in separate reactions)
  • Measuring synthesis yield using (14C) protein labeling and TCA precipitation
  • Characterization of protein expression using gel electrophoresis, autoradiography and quantitative imaging in the phosphorimager
  • Protein analysis using fluorescence microscopy and western blotting
  • Directed protein evolution using mutagenesis and activity screening
  • Cotranslational labeling of antibody fragments, e.g. with fluorescent dyes
  • Functional tests on cell-free synthesized antibody fragments, e.g. with ELISA
  • Optimization of in-vitro translation systems for the synthesis of disulfide-bridged proteins

S1 safety class cell culture laboratories

  • 5 L fermenter (Sartorius Biostat B DCU-II Advanced Additive Flow System; 2 x 5 L vessels, expandable up to 6 vessels between 1 and 10 L)
  • 30 L fermenter (Sartorius Biostat D DCU)
  • Confocal scanning laser microscope (Zeiss CLSM 510)
  • Automated cell assay and screening unit (PerkinElmer; CellLuxCellularFluorescence Workstation)
     

Isotope laboratory

  • Protein labeling (handling with 14C, 32P, 35S)
  • Extraction unit for the separation of 14C-labeled protein precipitates (TCA precipitation)
  • Scintillation counter (Beckmann LS 6500 Multi-Purpose Scintillation Counter)
  • Gel drying unit for autoradiograms (Unigeldryer 3545)
  • Typhoon Trio+ Variable-Mode Imager (radioactivity, fluorescence and chemiluminescence with advanced 10 µm pixel scan)
     

S1 safety class laboratory for molecular biological work

  • Multimode Reader Berthold LB 941 vi-S TriStar (flash, glow and color luminescence, absorption, fluorescence, FRET, BRET)
  • Sirius single tube luminometer (Titertek Berthold)
  • Spectrophotometer for UV/Vis (Nanodrop ND-2000c)
  • Bioreactors for cell-free protein synthesis using batch process and on dialysis scale
     

Mass spectrometry in S1 safety class laboratories

  • Mass spectrometer Q-TOF maXis Impact (Bruker Daltonics) with interchangeable ion sources (offline nanoESI source, conventional ESI source, captive spray for Nano-LC coupling)
  • Ultra-sensitive ion trap AmaZon Speed ETD (Bruker Daltonics) with interchangeable ion sources (conventional ESI source, captive spray for the nano-LC coupling)
  • UHPLC chromatography systems, Ultimate 3000 nanoRSLC system (Dionex)


Analysis of membrane proteins in S1 safety class laboratories

  • Port-A-Patch patch-clamp system and Orbit 16 made by Nanion
  • Particle analysis with Zetasizer Nano ZS made by Malvern

Publications

  • Jérôme V, Thoring L, Salzig D, Kubick S, Freitag R. Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor. Engineering in Life Sciences 2017 Aug 7. doi:10.1002/elsc.201700005
  • Zemella A, Grossmann S, Sachse R, Sonnabend A, Schaefer M, Kubick S. Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses. Sci Rep. 2017 Jun 16;7(1):3740. doi:10.1038/s41598-017-03955-8Artikel
  • Georgi V, Georgi L, Blechert M, Bergmeister M, Zwanzig M, Wüstenhagen DA, Bier FF, Junga E, Kubick S. On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode. Lab Chip. 2016 Jan 5;16(2):269-81. DOI dx.doi.org/10.1039/c5lc00700c. Artikel
  • Quast RB, Ballion B, Stech M, Sonnabend A, Varga BR, Wüstenhagen DA, Kele P, Schiller SM, Kubick S. Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids. Sci Rep. 2016 Sep 27;6:34048. DOI dx.doi.org/10.1038/srep34048 Artikel
  • Quast RB, Sonnabend A, Stech M, Wüstenhagen DA, Kubick S. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format. Sci Rep. 2016 Jul 26;6:30399. DOI dx.doi.org/10.1038/srep30399 Artikel
  • Thoring L, Wüstenhagen DA, Borowiak M, Stech M, Sonnabend A, Kubick S. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of »Difficult-to-Express« Proteins and Future Perspectives. PLoS One. 2016 Sep 29;11(9):e0163670. DOI dx.doi.org/10.1371/journal.pone.0163670 Artikel
  • Zemella A, Thoring L, Hoffmeister C, Kubick S. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem. 2015 Oct 19. DOI dx.doi.org/10.1002/cbic.201500340. [Epub ahead of print] Artikel
  • Quast RB, Mrusek D, Hoffmeister C, Sonnabend A, Kubick S. Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis. FEBS Lett. 2015 Jul 8;589(15):1703-12. DOI dx.doi.org/10.1016/j.febslet.2015.04.041. Epub 2015 May 1. Artikel
  • Bechlars S, Jäckel C, Diescher S, Wüstenhagen DA, Kubick S, Dieckmann R, Strauch E. Characterization of trh2 Harbouring Vibrio parahaemolyticus Strains Isolated in Germany. PLOS ONE | DOI dx.doi.org/10.1371/journal.pone.0118559. Artikel
  • Quast RB, Kortt O, Henkel J, Srujan KD, Wüstenhagen DA, Stech M, Kubick S. Automated production of functional membrane proteins usingeukaryotic cell-free translation systems. Journal of Biotechnology 203 (2015) 45–53. Artikel
  • Brödel AK, Wüstenhagen DA, Kubick S. Cell-Free Protein Synthesis Systems Derived from Cultured Mammalian Cells. Methods Mol Biol. 2015;1261:129-40. doi: 10.1007/978-1-4939-2230-7_7.
  • Stech M, Kubick K. Cell-Free Synthesis Meets Antibody Production: A Review. Antibodies 2015, 4, 12-33; DOI dx.doi.org/10.3390/antib4010012.
  • Dondapati SK, Kreir M, Quast RB, Wüstenhagen DA, Brüggemann A, Fertig N, Kubick S. Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system. Biosens Bioelectron. 2014 Sep 15;59:174-83.
  • Sachse R, Dondapati SK, Fenz SF, Schmidt T, Kubick S. Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes. FEBS Letter. 2014 Aug 25;588(17):2774-81. DOI dx.doi.org/10.1016/j.febslet.2014.06.007
  • Stech M, Hust M, Schulze C, Dübel S, Kubick S. Cell-free eukaryotic systems for the production, engineering, and modification of scFv antibody fragments. Engineering in Life Sciences. 2014;14(4):387–398. DOI dx.doi.org/10.1002/elsc.201400036.
  • Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S. A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells. PLoS One 9 (2014) e96635.
  • Scheller FW, Yarman A, Bachmann T, Hirsch T, Kubick S, Renneberg R, Schumacher S, Wollenberger U, Teller C, Bier FF. Future of biosensors: a personal view. Adv Biochem Eng Biotechnol. 2014;140:1-28.
  • Stech M, Brödel AK, Quast RB, Sachse R, Kubick S. Cell-free systems: functional modules for synthetic and chemical biology. Advances in Biochemical Engineering/Biotechnology, Springer Berlin Heidelberg (2013) 67-102.
  • Quast RB, Claussnitzer I, Merk H, Kubick S, Gerrits M. Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems. Anal Biochem. 2014 Apr 15;451:4-9.
  • Fenz SF, Sachse R, Schmidt T, Kubick S. Cell-free synthesis of membrane proteins: tailored cell models out of microsomes. Biochimica et Biophysica Acta. 2014 May;1838(5):1382-8. DOI dx.doi.org/10.1016/j.bbamem.2013.12.009.
  • Sachse R, Wüstenhagen D, Samal?kova M, Gerrits M, Bier FF, Kubick S. Synthesis of membrane proteins in eukaryotic cell-free systems. Eng. Life Sci. 2013, 13, No. 1, 39–48.
  • Bechlars S, Wüstenhagen DA, Drägert K, Dieckmann R, Strauch E, Kubick S. Cell-free synthesis of functional thermostable direct hemolysins of Vibrio parahaemolyticus. Toxicon. 2013 Dec 15;76:132-42.
  • Brödel AK, Sonnabend A, Kubick S. Cell-free protein expression based on extracts from CHO cells. Biotechnol Bioeng. 2014 Jan;111(1):25-36.
  • Brödel AK, Sonnabend A, Roberts L, Stech M, Wüstenhagen DA, Kubick S. IRES-Mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems. PLoS One 8 (2013) e82234.
  • Brödel AK, Raymond JA, Duman JG, Bier FF, Kubick S. Functional evaluation of candidate ice structuring proteins using cell-free expression systems. J Biotechnol. 2013 Feb 10;163(3):301-10.
  • Stech M, Merk H, Schenk JA, Stöcklein W, Wüstenhagen DA, Micheel B, Duschl C, Bier FF, Kubick S. Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system. J Biotechnol. 164 (2012) 220-231.
  • Zampatis DE, Rutz C, Furkert J, Schmidt A, Wüstenhagen D, Kubick S, Tsopanoglou NE, Schülein R. The protease-activated receptor 1 possesses a functional and cleavable signal peptide which is necessary for receptor expression. FEBS Lett. 2012 Jul 30;586(16):2351-9.
  • Orth JH, Schorch B, Boundy S, Ffrench-Constant R, Kubick S, Aktories K. Cell-free synthesis and characterization of a novel cytotoxic pierisin-like protein from the cabbage butterfly Pieris rapae. Toxicon. 2011 Feb;57(2):199-207.
  • Shaklee PM, Semrau S, Malkus M, Kubick S, Dogterom M, Schmidt T. Protein incorporation in giant lipid vesicles under physiological conditions. Chembiochem. 2010 Jan 25;11(2):175-9.
  • Royall E, Woolaway KE, Schacherl J, Kubick S, Belsham GJ, Roberts LO. The Rhopalosiphum padi virus 5' internal ribosome entry site is functional in Spodoptera frugiperda 21 cells and in their cell-free lysates: implications for the baculovirus expression system. J Gen Virol. 2004 Jun;85(Pt 6):1565-9.

 

Patents

  • STECH, Marlitt; HANACK, Katja; MESSERSCHMIDT, Katrin; KUBICK, Stefan: METHOD FOR PRODUCING POLYCLONAL ANTIBODIES USING AN ANTIGENIC COMPOSITION COMPRISING PROTEIN-CONTAINING MEMBRANE VESICLES. PCT/EP2014/002592
  • STECH, MARLITT; QUAST, ROBERT; WUESTENHAGEN, DOREEN; KUBICK, STEFAN: Verfahren und vorrichtung zur zellfreien proteinsynthese in gegenwart eines caspase-inhibitors. PCT/EP2014/002520