Cell-free Protein Synthesis

The Cell-free Protein Synthesis Unit is engaged in the synthesis of recombinant proteins in various cell-free systems. Special focus is on the characterisation, modification and functional examination of cell-free manufactured antibody formats. In order to analyse proteins, they must first be provided in a functionally active form. The expression of proteins in living cells, i.e. in vivo, is frequently used but does not always lead to the desired goal, since not every protein in cell cultures can be satisfactorily synthesized. An efficient alternative to the expression of proteins in living cells is cell-free protein synthesis. In this procedure, the constituents of the cell are used to produce a certain target protein in a rapid and cost-effective manner. A special advantage of using eukaryotic cell lysates is that they permit the synthesis of proteins with post-translational modifications (PTMs).

  • Manufacture, 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 manufacture of antibody formats in various reaction procedures, e.g. in the batch and dialysis mode or coupled (transcription and translation in one reaction system) and decoupled (transcription and translation in separate reactions)
  • Determination of the synthesis yield by means of (14C) protein labelling and TCA precipitation
  • Characterization of the protein expression by gel electrophoresis, autoradiography and quantitative imaging by phosphorus imager
  • Protein analysis by means of fluorescence microscopy and Western blot
  • Aligned protein evolution by mutagenesis and activity screening
  • Co-translational marking of antibody fragments, e.g. with fluorescence dyes
  • Functional investigations on cell-free manufactured antibody fragments, e.g. by ELISA
  • Optimization of in-vitro translation systems for disulphide bridged synthesis proteins

Cell-Free Antibody Synthesis

Specific antibodies are of inestimable value both in diagnostics as well as therapeutics. Therefore cell-free protein synthesis systems based on eukaryotic cell extracts are used as a new strategy for efficient and reliable synthesis and selection of suitable antibody candidates. Enormous time savings and cost efficiency can be achieved compared to conventional methods of generating antibody fragments. Furthermore, open eukaryotic in-vitro translation systems provide great leeway for modifications and improvements in the quality of characteristics of cell-free manufactured proteins. One potentially pharmacologically-relevant result, for example, is offered by the cell-free synthesis of antibody-active ingredient conjugates that cannot be synthesized in vivo. By optimising the biological process steps in defined reaction environments, the synthesis conditions can be individually adapted to the requirements of the respective proteins. Parallel expression screening of different protein-coded sequences is possible in this way, as well as testing suitable reaction parameters for a defined protein.

Cell Culture Laboratories of Safety Class S1

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

Isotope Laboratory

  • Protein labeling (interaction with 14C, 32P, 35S)
  • Exhaust systems for the separation of 14C labeled protein precipitates (TCA precipitation)
  • Scintillation counters (6500 Multi)
  • Gel drying unit for autoradiographs (Unigeldryer 3545)
  • Typhoon Trio+ variable-mode imager (radioactivity, fluorescence and chemiluminescence with extended 10 µm pixel scan)

Laboratories of the Safety Class S1 for Molecular-Biological Work

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

Mass Spectrometry in Laboratories of Safety Class S1

  • 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, 3000 system (Dionex)

Analytics of Membrane Proteins in Laboratories of Safety Class S1

  • Patch-Clamp system "Port-A-Patc" and Orbit 16 from Nanion
  • Particle analysis by means of “Zetasizer Nano ZS” from Malvern

  • 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. Article
  • 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 Article
  • 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 Article
  • 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 Article
  • 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] Article
  • 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. Article
  • 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. Article
  • 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. Article
  • 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.