JIOMICS

JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62
JOURNAL OF INTEGRATED OMICS
Journal of Integrated Omics
A METHODOLOGICAL JOURNAL
HTTP://WWW.JIOMICS.COM
Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015)
Cell-free lysates for the production of difficult proteins
F. Bernhard* 1 , D. Foshag 2 , S. Rupp 3 , Ernst Thuer 4 , V. Dötsch 1 , E. Henrich 1 , R. Rues 1
1
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt-am-Main, Germany.
2
Institute for Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Stuttgart, Germany. 3 Fraunhofer Institute for
Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany. 4 Comparative Genomics Group, CRG-Centre for Genomic Regulation,
Barcelona, Spain. *Corresponding author: fbern@bpc.uni-frankfurt.de
Available Online: 31 December 2015
Abstract
Purpose: Cell-free production of proteins and other biomolecules has emerged during the last decade as an important core technology in the
new field of synthetic biology. Lysates of either eukaryotic or prokaryotic organisms can be employed for the synthesis of particularly difficult
proteins such as membrane proteins, large multisubunit assemblies or highly oxidized proteins. Cell-free expression provides an open and
accessible reaction environment. Additionally, functional properties of the lysate proteomes can easily be modulated in order to generate ideal
conditions for the production of high quality samples of individual target proteins.
Experimental description: Protocols for the preparation of cell-free lysates have been developed and optimized. Libraries of additives have
been screened for the manipulation and optimization of lysate proteomes.
Results: We present strategies for the manipulation of cell-free lysates and reaction conditions for the efficient production of difficult proteins
such as G-protein coupled receptors or enzymes. We further exemplify the optimization of protein sample quality by directed fractionation
of lysate proteomes and selective inactivation of particular enzyme classes and pathways. Examples focusing on the structural and functional
characterization of membrane proteins will be discussed.
Conclusions: Lysate proteomes for the cell-free production of biomolecules can be adjusted according to the requirements of individual targets.
Keywords: cell-free expression, bacterial lysates, membrane protein environments, synthetic biology, artificial proteomes.
Acknowledgements: The work was funded by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG)
and by the German Ministry of Education and Science (BMBF). Support was further obtained by Instruct, part of the European Strategy Forum
on Research Infrastructures (ESFRI).
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