Cell-free protein expression

Cell-free lysates are a useful alternative to the expression of recombinant proteins in living cells. They are generated by lysing eucaryotic or bacterial cells and removal of all components that are not required for protein expression. These lysates can then be combined with suitable DNA templates and other required components in reaction tubes to start protein expression (1).
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Advantages

1. Cell-free reactions are easy to set up and take only several hours to express protein. This way, optimization of reaction conditions can be done fast in parallel using small scale (50-100 µl) reactions. Optimal conditions can be linearly scaled up to the required amounts.
2. Overall handling volumes are small: for soluble proteins, mg amounts of protein can be obtained from few ml reaction volumes.
3. As an open system, many components - natural and unnatural - can be added to the reaction mixture, such as reducing/oxidizing elements, chaperones, labeled amino acids, or detergents. This way, proteins can be directly used in applications like NMR, and also membrane proteins can be reconstituted into nanodisc systems in their nascent state, without any artificial effect of detergents (10-12).
4. Eucaryotic cell-free lysates provide post-translational modifications such as core glycosylation or phosphorylation, and natural membrane components (e.g. microsomes) for membrane protein insertion (9).
5. Proteins that are toxic when expressed in living cells, can often be produced in cell-free systems. Also other effects like protease digest or DNA template instability are absent because the responsible cellular components have been removed.
6. Multimeric proteins and complexes can be expressed, and ratios of different subunits influenced by DNA template titration.

Disadvantages

1. Cell-free expression can become costly if large amounts of protein are required, or for proteins that do not express well. Therefore for each protein, achievable yields should be assessed in small scale experiments and cost for upscales calculated. This is especially true for more complex cell-free lysates from eucaryotic sources.
 E.coliWheat germInsectMammalian
Yield (relative) high (several mg/ml) medium medium-low low
Cost (relative) low medium medium high
Phosphorylation no yes yes yes
Glycosylation no core core mammalian
Membrane protein options insoluble detergent nanodisc liposomes detergent liposomes natural endosomes detergent liposomes nanodiscs natural microsomes detergent liposomes
Commercially available batch mastermix CECF kits individual cell lysates batch & CECF kits batch mastermix batch mastermix
Cube Biotech offering E.coli cell lysates for batch and CECF nanodiscs detergents      
Table 1. Comparison of different cell-free expression systems.

Applications

Because proteins are expressed in an open system, there are many applications that are facilitated by cell-free expression in comparison to expression in living cells. These include:
Expression screening: With cell-free expression, you can go from DNA template - or even multiple reactions in parallel - to Western Blot result in two days, making the system attractive for fast screening of multiple constructs and reaction conditions.
Protein labelling: The open system can be combined with labeled amino acids to introduce e.g. biotin or fluorescent labels.
NMR: Isotope-labeled amino acids can be easily added to the mix to obtain proteins ready for NMR.
Crystallization: In combination with selenomethionine, protein heavy metal derivatives are obtained in a straightforward procedure.

Special adaptations for membrane proteins

Because proteins are expressed in an open system, there are many applications that are facilitated by cell-free expression in comparison to expression in living cells. These include:
insoluble expression: without the addition of any kind of lipid or detergent, membrane proteins form aggregates that in many cases can be refolded into functional proteins.
detergent: Detergents, especially of the Brij series, have been added in many cases to solubilize membrane proteins. If needed, a detergent exchange can be performed in subsequent steps, preferably during purification. (2-7)
nanodisc: Adding nanodiscs to cell-free reactions is the most simple and elegant way to obtain stabilized proteins that can be used for a variety of assays without the need for detergent (10-15). Applications range from biophysical assays, SPR, NMR, mass spectrometry to cryo-EM. Recently, there is evidence that membrane proteins expressed in cell-free lysates in the presence of nanodiscs can be crystallized by in meso methods (14). Learn more about nanodiscs.
liposomes and other lipid particles: Liposomes, microsomes and other lipid-containing structures such as bicelles have been added to cell-free reactions, in particular in combination with insect cell lysates, for membrane protein expression (9). Using appropriate detergents, membrane proteins can be shifted from nanodiscs into bicelles (13).

Batch vs CECF methods

Cell-free reactions can be performed in two general procedures:
Batch: Batch cell-free reactions are very easy to set up in simple vials like microcentrifuge tubes. Reaction times are short (1-3 hours) because energy components are used up quickly, and accumulation of end products slows down the enzymatic reactions. Therefore, protein yields are typically low.
CECF or dialysis: Continuous exchange cell-free (CECF) reactions are set up in dialysis chambers, so that energy components are fed into the reaction, and end products such as ADP are removed. This way, reaction times up to 24 hours are possible, and protein yields obtained can reach several mg per ml reaction for well-expressing soluble proteins (8).
Literature references:
1. Endo, Y., et al. (eds) Cell-Free Protein Production, Methods and Protocols. Methods in Molecular Biology vol. 607, 2010
2. Boland, C. et al. Cell-free expression an in meso crystallization of an integral membrane kinase for structure determination. Cell. Mol. Life Sci (2014) DOI: 10.1007//s00018-014-1655-7
3. Hein, C. et al. Hydrophobic environments in cell-free systems: Designing artificial environments for membrane proteins. J. Eng. Life Sci. (2014), 14, 365-79.
4. Proverbio, D., et al. Membrane protein quality control in cell-free expression sysems: Tools, strategies and case studies. Membrane proteins production for structural analysis (Isabelle Mus-Veteau, ed.) Springer Heidelberg. ISBN:978-1-4939-0662-8.
5. Haberstock, S. et al. A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems. Prot. Exp. Purific. (2012) 82, 308-16.
6. Matthies, D, et al. Cell-free expression and assembly of a macromolecular membrane protein complex. J. Mol. Biol. (2011) 413, 593-603.
7. Schneider, B. et al. Membrane protein expression in cell-free systems. Metho. Mol. Biol. (2010) 601, 165-86.
8. Schwarz, D. et al. Preparative scale expression of membrane proteins in E.coli based continuous exchange cell-free systems. Nat. Protocols (2007) 2, 2945-57.
9. Sachse, R. et al. Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes. FEBS Lett. (2014), 588,2774-2781
10. Roos, C. et al. Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E.coli MraY translocase. Biochim. Biophys. Acta (2012) 1818; 3098-3106
11. Proverbio, D. et al. Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim. Biophys. Acta (2013) 1828, 2182-92.
12. Roos, C. et al. High-level cell-free production of membrane proteins with nanodiscs. In: Cell-free Protein Synthesis: Methods and Protocols. Alexandrov, K., and Johnston, W.A. (eds), Methods in Molecular Biology (2014), vol. 1118, Chapter 7.
13. Laguerre, A. et al. From nanodiscs to isotropic bicelles: A procedure for solution nuclear magnetic resonance studies of detergent-sensitive integral membrane proteins. Structure (2016) 24, 1-12.
14. Nikolaev, M. et al. Integral membrane proteins can be crystallized directly from nanodiscs. Cryst. Growth Des. (2017) 17(3), 945–948
15. Henrich, E., et al. Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology. eLife (2017) 6:e20954.
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