- Cell-free expression platform at the IBPC
- Cell-free membrane protein production
- Co-translational insertion of membrane proteins into nanoparticles
- Characterization of cell-free sythesized GPCR/G-protein complexes
- Nanomembranes as transfer vectors for integrated membrane proteins
- Cell-free labelling of proteins
1. Cell-free expression platform at the IBPC
We use cell-free expression technology for the production of challenging proteins such as membrane proteins or toxins, as well as for the labelling of proteins with stable isotopes or fluorescent markers.
Our established in-house cell-free expression system comprises the production of cell-free lysates from special E. coli strains, the manufacturing of reaction containers, and the preparation of additives such as nanoparticles or enzymes.
Our lysates are standardized and characterized by proteome analysis (Foshag 2018).
Most efficient with > 3 mg/mL protein production is the HY-S30 lysate (available at CUBE-Biotech).
The HS-S30 lysate is enriched in chaperones suitable for optimized protein folding.
- Foshag D et al (2018) The E. coli S30 lysate proteome: Prototype for cell-free synthetic biology. New Biotechnol 40: 245-60.
- Kai L et al (2015) Co-translational stabilization of insoluble proteins in cell-free expression systems. Meth Mol Biol 1258: 125-43.
- Kai L et al (2013) Artificial environments for the co-translational stabilization of cell-free expressed proteins. PlosOne 8, e56637.
- Bernhard F et al (2013) Cell-free expression – making a mark. Cur Opin Struct Biol 23: 374-80.
- Haberstock S et al (2012) A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems. Prot Expr Purific 82: 308-16.
- Kai L et al (2011) Systems for the cell-free synthesis of proteins. Meth Mol Biol 800: 201-25.
- Junge F et al (2010) Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins. New Biotechnol 28: 262-71.
- Schwarz D et al (2007) Preparative scale expression of membrane
2. Cell-free membrane-protein production
Membrane proteins can be cell-free synthesized in three different basic modes. Which mode is most suitable for a given membrane protein is subject of screening.
Structures of cell-free synthesized membrane proteins: Different views of D-CF synthesized proteorhodopsin (Reckel 2011), P-CF synthesized Diacyl-glycero-kinaseA (Boland et al., 2014), fully assembled D-CF synthesized ATP synthase (Matthies et al., 2011).
- Mezhyrova J et al (2022) Applications of cell-free synthesized membrane protein precipitates. Meth Mol Biol 2406: 245-66.
- Mezhyrova J et al (2021). Membrane insertion mechanism and molecular assembly of the bacteriophage lysis toxin ΦX174-E. FEBS J 288: 3300-16.
- Keller T et al (2019) Rat organic cation transporter 1 contains three substrate binding sites per monomer. Mol Pharmacol. 95: 169-82.
- Henrich E et al (2018) Lipid conversion by cell-free synthesized phospholipid methyltransferase Opi3 in defined nanodisc membranes supports in trans mechanism. Biochemistry 57: 5780-4.
- Waberer, L et al (2018) The synaptic vesicle protein SV31 assembles into a dimer and transports Zn2+. J Neurochem DOI: 10.1111/jnc.13886.
- Focke P et al (2016) Combining in vitro folding with cell-free protein synthesis for membrane protein expression. Biochemistry 55:4212-9.
- Rues RB et al (2016) Cell-free production of membrane proteins in Escherichia coli lysates for functional and structural studies. Meth Mol Biol 1432: 1-22.
- Hein C et al (2014) Hydrophobic environments in cell-free systems: Designing artificial environments for membrane proteins. J Eng Life Sci 14: 365-79.
- Merk H et al (2015) Biosynthesis of translocation dependent proteins in insect cell lysates: Identification of key parameters for folding and processing. Biol Chem 396: 1097-107.
- Boland C et al (2014) Cell-free expression and in meso crystallization of an integral membrane kinase for structure determination. Cell Mol Life Sci 71: 4895-910.
- Reckel S et al (2011) Solution structure of proteorhodopsin. Angew Chem Intl Ed DOI: 10.1002/anie.201105648.
- Matthies D et al (2011) Cell-free expression and assembly of a macromolecular membrane protein complex. J Mol Biol 413: 593-603
- Ma Y et al (2011) Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes. J Biol Chem 286: 38844-53.
- Schwarz D et al (2010) Cell-free expression profiling of E. coli inner membrane proteins. Proteomics 10: 1762-79.
3. Co-translational insertion of membrane proteins into nanoparticles
A special L-CF strategy only possible by cell-free expression is the direct cotranslational insertion of nascent membrane proteins into provided nanomembranes. The insertion is translocon independent and avoids any contact with detergents. The resulting membrane protein/nanoparticle complexes can instantly be used for structural or functional studies.
Nanodiscs can be loaded with almost any lipid combination and allow the systematic screening of lipid effects on protein function (Henrich et al., 2015)
Three nanoparticle formation strategies are possible with nanodiscs or Salipro particles:
- 1: Preforming;
- 2: Coforming;
- 3: Coexpression
(Levin et al, in revision)
- Grethen A et al. Electroneutral polymer nanodiscs enable interference-free probing of membrane proteins in a lipid-bilayer environment. Small, in revision.
- Levin R et al. Cotranslational assembly of membrane protein/nanoparticles in cell-free systems. Biochim Biophys Acta, in revision.
- Levin R et al (2020). Co-translational insertion of membrane proteins into preformed nanodiscs. J Vis Exp 165: e61844.
- Peetz O et al (2017) Insights into co-translational membrane protein insertion by combined LILBID-mass spectrometry and NMR spectroscopy. Anal Chem 89: 12314-8.
- Rues RB et al (2017) Membrane protein production in E. coli lysates in presence of preassembled nanodiscs. Meth Mol Biol 1586:291-312.
- Henrich E et al (2015) Membrane protein production in Escherichia coli cell-free lysates. FEBS Lett 589: 1713-22.
- Henrich E et al (2017) Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology. eLife 6:e20954.
- Henrich E et al (2016) Lipid requirements for the enzymatic activity of MraY translocase homologues and in vitro reconstitution of Lipid II synthesis pathway. J Biol Chem 291: 2535-46.
- Henrich E et al (2015) Screening for lipid requirements of membrane proteins by combining cell-free expression with nanodiscs. Meth Enzymol 556: 351-69.
- Mörs K et al (2013) Modified lipid and protein dynamics in nanodiscs. Biochim Biophys Acta 1828: 1222-9.
- Roos C et al (2012) Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase. Biochim Biophys Acta 1818: 3098-106.
- Zocher M et al (2011) Single-molecule force spectroscopy from nanodiscs: An assay to quantify folding, stability, and interactions on native membrane proteins. ACS Nano 6: 961-71.
4. Characterization of cell-free synthesized GPCR/G-protein complexes
G-protein coupled receptors are synthesized by cotranslational insertion into tailored nanodisc membranes without any contact to detergents. The GPCRs are full-length and functional in ligand binding and G-protein coupling.
Negative stain and cryo-electron microscopy of cell-free synthesized GPCR/G-protein complexes in nanodiscs
(Köck et al., 2022)
- Köck Z et al (2022). Biochemical characterization of cell-free synthesized human β1 adrenergic receptor cotranslationally inserted into nanodiscs. J Mol Biol434:167687. doi:10.1016/j.jmb.2022.167687.
- Köck Z et al (2021) Screening methods for cell-free synthesised GPCR/nanoparticle samples. Meth Mol Biol 2268: 97-117.
- Krug U et al (2020) The conformational equilibrium of the neuropeptide Y2 receptor. Angew Chem Int Ed 59:2-10.
- Pacull EM et al (2020) Integration of cell-free expression and solid-state NMR to investigate the dynamic properties of different sites of the growth hormone Secretagogue receptor. Front Pharmacol 11:562113.
- Dong F et al (2018) Molecular determinants for ligand selectivity of the cell-free synthesized human endothelin B receptor. J Mol Biol 430: 5105-5119.
- Rues RB et al (2018) Systematic optimization of cell-free synthesized human endothelin B receptor folding. Methods 147: 73-83.
- Rues RB et al (2016) Co-translational formation and pharmacological characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments. Biochim Biophys Acta 1858: 1306-16.
- Orbán E et al (2015) Cell-free expression of G-protein coupled receptors. Meth Mol Biol 1261: 171-95.
- Rues RB et al (2014) Cell-free expression of G-protein coupled receptors: New pipelines for challenging targets. Biol Chem 395: 1425-34.
- Proverbio D (2013) Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim Biophys Acta 1828: 2182-92.
5. Nanomembranes as transfer vectors for integrated membrane proteins
Membranes of nanodiscs or Salipro particles efficiently fuse with other membranes and inserted membrane proteins can thus even be transferred into membranes of living cells (Patriarchi et al, Sci Rep). This new technique allows simultaneous in vitro and in vivo assays with same sample batch.
Transfer of the ion pump KR2 from nanodiscs into solid supported membranes (Henrich 2017) and bacteriorhodopsin from nanodiscs into lipidic cubic phase for subsequent crystallization (Nikolaev et al., 2017).
Localization of transferred GPCR-mNG compared with endogenuous synthesized GPCR-mNG after transfection. Transferred GPCRs are active in ligand binding, G-protein coupling and interact with endogenuous binding partners.
(Umbach et al., 2022)
- Umbach S et al (2022). Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells. Front Bioeng Biotechnol 10:906295. doi:10.3389/fbioe.2022.906295.
- Henrich E et al (2017) From gene to function: Cell-free electrophysiological and optical analysis of ion pumps in nanodiscs. Biophys J 113: 1331-41.
- Nikolaev M et al (2017) Integral membrane proteins can be crystallized directly from nanodiscs. Cryst Growth Des 17: 945-8.
6. Cell-free labelling of proteins
Cell-free expression gives complete control over the amino acid pool in the reaction. It is therefore the most efficient system for fast labelling of proteins, either by full-labelling or by combinatorial-labelling strategies.
Combinatorial labelling of the putative multidrug transporter TehA (Trbovic et al., 2005).
Reduction of NMR spectral complexity by methyl labelling of amino acid residues, exemplified with the proton pump proteorhodopsin (Lazarova et al., 2018).
- Henrich E et al (2019) Synthetic biology based solution NMR studies on membrane proteins in lipid environments. Methods Enzym 614: 143-85.
- Lazarova M et al (2018) Precursor-based selective methyl labelling of cell-free synthesized proteins. ACS Chem Biol 13: 2170-78.
- Hoffmann B et al (2018) Protein labeling strategies for liquid-state NMR spectroscopy using cell-free synthesis. Progress in NMR spectroscopy 105: 1-22.
- Laguerre A et al (2016) From nanodiscs to isotropic bicelles: A procedure for solution nuclear magnetic resonance studies of detergent-sensitive integral membrane proteins, Structure 24: 1-12.
- Laguerre A et al (2015) Labeling of membrane proteins by cell-free expression. Meth Enzymol 565: 367-88.
- Tumulka F et al (2013) Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR. J Biomol NMR 57: 141-54.
- Löhr F et al (2012) Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment. J Biomol NMR 52:197-210.
- Sobhanifar S et al (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J BioMol NMR 46: 33-43.
- Reckel S et al (2008) Transmembrane segment enhanced labeling as a tool for the backbone assignment of a-helical membrane proteins. Proc. Natl. Acad. Sci. USA 105: 8262-7.
- Trbovic N et al (2005) Efficient strategy for the rapid backbone assignment of membrane proteins. J Am Chem Soc 127: 13504-5.