Cell-Free Systems: Functional Modules for Synthetic and Chemical Biology

  • Marlitt Stech
  • Andreas K. Brödel
  • Robert B. Quast
  • Rita Sachse
  • Stefan KubickEmail author
Part of the Advances in Biochemical Engineering/Biotechnology book series (ABE, volume 137)


The main goal of cell-free protein synthesis is to produce correctly folded and functional proteins in reasonable amounts for further downstream applications. Especially for eukaryotic proteins, functionality is often directly linked to the presence of posttranslational modifications. Thus, it is of highest interest to develop novel cell-free expression systems that enable the synthesis of posttranslationally modified proteins. Here we present recent advances for the synthesis of glycoproteins, proteins containing disulfide bridges, membrane proteins, and fluorescently labeled proteins. The basis for the expression of these difficult-to-express target proteins is a translationally active cell extract which can be prepared from eukaryotic cell lines such as Spodoptera frugiperda 21 (Sf21) and Chinese hamster ovary (CHO) cells. Due to a very mild lysate preparation procedure, microsomal vesicles derived from the endoplasmic reticulum (ER) can be maintained in the eukaryotic lysate. These vesicles are translocationally active and serve as functional modules facilitating protein translocation and enrichment as well as posttranslational modification of de novo synthesized proteins. In particular, for the synthesis of membrane proteins microsomal vesicles are the essential prerequisite for the insertion of the desired protein into a biologically active membrane scaffold providing a natural environment. We anticipate that the use of such translationally active eukaryotic cell lysates containing translocationally active vesicles may solve a large number of problems still persistent when expressing eukaryotic proteins in vitro.

Graphical Abstract


Cell-free protein synthesis Fluorescent label GUV In vitro translation Insect cell lysate Membrane protein Microsome Noncanonical amino acid Orthogonal tRNA/Synthetase pair Posttranslational modification Single-chain antibody (scFv) Vesicle 







Orthogonal mutant synthetase specific for AzPhe


Bandpass filter


Continuous-exchange cell-free


Continuous-flow cell-free


Chinese hamster ovary


Confocal laser scanning microscopy


Cricket paralysis virus





E. coli

Escherichia coli


Endoglycosidase H






Endoplasmic reticulum


Enhanced yellow fluorescent protein


Fragment, antigen-binding


Fluorescein isothiocyanate


Antibody variable fragment




Giant unilamellar vesicle


Intergenic region


Internal ribosome entry site


Association equilibrium constant




Polyacrylamide gel electrophoresis


Protein disulfide isomerase


Protease inhibitors


N-glycosidase F


Posttranslational modification


Resonance units


Single-chain antibody fragment


Sodium dodecyl sulfate


Spodoptera frugiperda 21


Supernatant 1


Supernatant 2


Surface plasmon resonance


Trichloroacetic acid


Translation mixture


Amber suppressor tRNA


Vesicular fraction 1


Vesicular fraction 2



The authors gratefully acknowledge Dipl.-Ing. Doreen Wüstenhagen, Dipl. Nutritional Scientist Conny Mascher, and Birgit Hollmann (Fraunhofer IBMT, Potsdam-Golm, Germany) for preparing cell-free extracts and in general, for keeping things running in our cell-free lab. Furthermore, we thank Dr. Walter Stöcklein (Fraunhofer IBMT, Potsdam-Golm, Germany) for his excellent support regarding SPR measurements as well as Prof. Burkhard Micheel (University of Potsdam, Germany) for providing the Anti-FITC monoclonal antibody. Moreover, we thank Jörg Schenk (Hybrotec GmbH, UP Transfer GmbH, Potsdam-Golm, Germany) for delivering the DNA template encoding the Anti-FITC scFv as well as for the scientific support and many fruitful discussions. Plasmids encoding the membrane proteins Hb-EGF, EGFR, and ETB were kindly provided by Prof. Michael Schaefer (Universität Leipzig, Germany). We also wish to express our considerable thanks to Dr. Susanne Fenz (Universität Würzburg, Germany) and Prof. Thomas Schmidt (Leiden University, The Netherlands) for their collaboration regarding the GVU formation process. Our special thanks go to Dipl.-Translator Marie Burger, Dipl.-Chem. Devid Mrusek, and Dipl.-Biochem. Christian Hoffmeister (Fraunhofer IBMT, Potsdam-Golm, Germany) for their careful revision of this manuscript. This research is supported by the German Ministry of Education and Research (BMBF No. 0312039).


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Marlitt Stech
    • 1
  • Andreas K. Brödel
    • 1
  • Robert B. Quast
    • 1
  • Rita Sachse
    • 1
  • Stefan Kubick
    • 1
    Email author
  1. 1.Fraunhofer IBMTBranch Potsdam, Group of Cell-free Protein SynthesisPotsdamGermany

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