E.coli cell lysates


Logo_Cellfree
For increased convenience and higher protein yields (up to 6 mg/ml GFP), the HiYield-T7 lysate was developed. It already includes the T7 RNA polymerase enzyme. As an alternative to protein expression in living cells, cell-free lysates can be used to obtain recombinant proteins. They are particularly useful for the following applications: Please also refer to our new HiYield-T7 lysates which already contain T7 RNA polymerase, which is required if a T7 promoter driven plasmid is used. For expression regulated by e.g. the tac promoter, addition of this enzyme is not required.

E.coli T7 lysates for cell-free expression from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
His-tagged Ups1 together with Mdm35 2019 Miliara X., Tatsuta T., Berry J-L., Rouse S.L.,Solak K., Chorev D.S., Wu D., Robinson C.V., Matthews S. 1
ETB receptor 2018 Rues R-B., Dong F., Dötsch V., & Bernhard F. 2
PRELID3B–TRIAP1 2019 MacVicar T., Ohba Y., Nolte H., Mayer F.-C., Tatsuta T., Sprenger H.-G., Lindner B., Zhao Y., Li J., Bruns C., Krüger M., Habich M., Rlemer J., Schwarzer R., Pasparakis M., Henscke S., Brüning J., Zamboni N., Langer T.2
 
Note that our E.coli lysates require additional components (e.g. amino acids, nucleic acids, DNA polymerase, dialysis devices) that are currently not provided by Cube Biotech. Please refer to the protocols for more details.
In close collaboration with the group of Volker Doetsch and Frank Bernhard, Institute of Biophysics at the University of Frankfurt, Germany, we provide an open system that can be adapted for a range of applications, with dedicated protocols for membrane proteins, continous exchange (dialysis) reactions, and many more. This system takes more work initially than ready to use kits, but provides ultimate flexiblity in reaction variations.
Hi-yield GFP Expression
Fig. 1: Standard vs. HiYield cell-free expression of GFP. 1,7: MW marker, 2-6: HiYield lysate expression; variation of Mg2+ concentration (14, 16, 18, 20,22 mM). 8-12: Standard lysate expression, same variation of Mg2+ concentrations. A Mg2+ optimum was observed at ca. 16 mM for these combinations of cell-free lysates and other reaction components. Yields range from 1.3-5 mg/ml for HiYield reactions, and from 1-3.8 mg/ml for standard reactions. Data kindly provided by Frank Bernhard, University of Frankfurt, Department of Biophysical Chemistry.

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Lyophilized MSP protein
Required equipment
 
  • Micropipettor and Micropipetting tips
  • Thermomix / shaking incubator for 500 rpm and 30°C incubation
  • Ultrasonic device (Optional)
  • Dialysis chambers and feeding mix reservoirs, depending on reaction scales. Alternatively home-built dialysis chambers combined with dialysis membrane, cutoff 12-14 kDa. See (1) for details.
Materials
 
  • E.coli lysate, for cell-free protein expression
  • 1,4-Dithiotreitol (DTT)
  • Acetyl phosphate lithium potassium salt (ACP)
  • Acetic Acid
  • Adenosine 5’-triphosphate (ATP)
  • Amino acids for cell-free expression
  • Complete protease inhibitor cocktail
  • Cytidine 5’-triphosphate di-sodium salt (CTP)
  • Dipotassium hydrogen phosphate (K2HPO4)
  • Ethylenediaminetetraacetic acid (EDTA)
  • Folinic acid calcium salt
  • Guanosine 5’-triphosphate di-sodium salt (GTP)
  • 2-(4-(2-Hydroxyethyl)- 1-piperazinyl)-ethane sulfonic acid (HEPES)
  • Magnesium acetate tetrahydrate (Mg(oAc)2)
  • Magnesium chloride hexahydrate (MgCl2)
  • Sodium azide (NaN3)
  • PEG 8,000
  • Phosphoenol pyruvic acid monopotassium salt (PEP)
  • Potassium acetate (KoAc)
  • Potassium chloride (KCl)
  • Potassium dihydrogenphosphate (KH2PO4)
  • Pyruvate kinase (PK)
  • RiboLock RNase inhibitor
  • Sodium chloride
  • Tris base
  • tRNA E.coli MRE 600
  • Uridine 5’-triphosphate tri-sodium salt (UTP)
  • Template DNA
  • Optional: Positive control template DNA
  • Recommended: Suitable affinity chromatography matrix or magnetic beads
  • SDS-PAGE/ Western Blot equipment and antibody
  • Potassium hydroxide (KOH)
  • Sodium hydroxide (NaOH)
 
For membrane protein expression:
  • Pre-assembled nanodisc
  • Detergents, e.g. Decylmaltoside, dodecylmaltoside

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
This protocol delineates all necessary steps, chemicals, and solutions to set up a cell-free protein expression reaction in a continuous exchange (dialysis) format. Compared to cell-free reactions in batch format, higher yields can be obtained in dialysis mode, because the reaction is continuously fed through the dialysis membrane, and reaction by-products are removed from the equilibrium. Reaction times can therefore be extended to overnight incubations. Cell-free reactions have vast opportunities because they can be supplemented with isotope labeled amino acids, fluorescence-labeled amino acids or other additives. For membrane protein expression, CECF reactions can be supplemented with detergents, nanodiscs, or other lipid-containing components such as bicelles or liposomes. The novel T7 lysates such as Cellfree E.coli lysates HiYield-T7 already contain the T7 RNA polymerase in optimal quantities. Therefore, this component can be omitted when using these lysates. Cell-free expression should first be optimized in analytical scale (50-100 µL) for parameters such as magnesium concentration, DNA template type and concentration. Optimal conditions can then be linearly scaled up to several mL volume. Please contact us if you have questions or need assistance optimizing a protocol for your application (contact@cube-biotech.com).
   
A.Setup of analytical scale CECF reactions
 
 
  1. Prepare all stock solutions as listed in the PDF-file, set pH with the given amounts of NaOH or KOH, sterile-filter, aliquot, and store.
  2. Prepare a DNA template coding for the protein of interest in a concentration of 0.2-0.5 mg/mL in ddH2O.
  3. Prepare a dialysis reaction container: either assemble a selfbuilt container with a dialysis membrane, or a commercially available reaction container according to the manufacturer’s instructions.
  4. Prepare the required amount of master mix for the number of reactions to be set up.
  5. Reconstitute reaction and feeding mixture in the required amounts.
  6. Check dialysis devices for leakage. Briefly incubate the dialysis membrane in the feeding mix before adding the reaction mix.
  7. Fill reaction and feeding mixture aliquots into reaction containers according to manufacturer’s instructions. When using home-built mini-CECF reactors, ensure that a fresh piece of dialysis membrane is used every time. Always make sure no air bubbles are trapped between the membrane and the solutions.
  8. When using a lysate batch and set of chemical stock solutions for the first time, perform a screen of Mg2+ and K+ concentrations using GFP as positive control. For optimization, screen different amounts of DNA template, and, optionally also concentrations of Mg2+ and K+.
  9. Incubate the CECF reactions overnight at 30°C with gentle shaking or rolling to ensure efficient substance exchange between reaction and feeding mixture.
  10. Spin the mixture at 15,000 rpm for at least 15 min to remove precipitates. Analyze both supernatants and precipitates in SDS-PAGE.
  11. Purify proteins from the reaction mixtures using appropriate affinity chromatography matrices.
  12. . Analyze protein in SDS-PAGE and, optionally, in a Western Blot using an appropriate antibody. For quantification of expressed protein via 35S-Methionine, refer to (1). Measure GFP fluorescence in GFP Assay buffer.

 
   
B.Setup of preparative scale CECF reactions
 
 
  1. After optimizing expression conditions for a given protein in analytical scale, calculate the reaction volume required to obtain the desired amount of recombinant protein.
  2. . Prepare enough DNA template coding for the protein of interest in a concentration of 0.2-0.5 mg/mL in ddH2O.
  3. Prepare a dialysis reaction container: either assemble a selfbuilt container with a dialysis membrane, or a commercially available reaction container according to the manufacturer’s instructions.
  4. Prepare the required amount of master mix for the number of reactions to be set up.
  5. Reconstitute reaction and feeding mixture in the required amounts.
  6. Fill reaction and feeding mixture aliquots into reaction containers according to manufacturer’s instructions.
  7. Incubate the CECF reactions overnight at 30°C with gentle shaking or rolling to ensure efficient substance exchange between reaction and feeding mixture.
  8. Purify proteins from the reaction mixtures using appropriate affinity chromatography matrices.
  9. Analyze protein in SDS-PAGE and, optionally, in a Western Blot using an appropriate antibody. For quantification of expressed protein via 35S-Methionine, refer to (3). Measure GFP fluorescence in GFP Assay buffer

References:

  1. Miliara, Xeni, et al. "Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins." Nature communications 10.1 (2019): 1130.
  2. Rues, Ralf-Bernhardt, et al. "Systematic optimization of cell-free synthesized human endothelin B receptor folding." Methods 147 (2018): 73-83.
  3. Roos, Christian, et al. "High-level Cell-free production of membrane proteins with Nanodiscs." Cell-Free Protein Synthesis. Humana Press, Totowa, NJ, 2014. 109-130.
  4. MacVicar, Thomas, et al. "Lipid signalling drives proteolytic rewiring of mitochondria by YME1L." Nature (2019): 1-5.
Logo_Cellfree
For increased convenience and higher protein yields (up to 6 mg/ml GFP), the HiYield-T7 lysate was developed. It already includes the T7 RNA polymerase enzyme. As an alternative to protein expression in living cells, cell-free lysates can be used to obtain recombinant proteins. They are particularly useful for the following applications: Please also refer to our new HiYield-T7 lysates which already contain T7 RNA polymerase, which is required if a T7 promoter driven plasmid is used. For expression regulated by e.g. the tac promoter, addition of this enzyme is not required.

E.coli T7 lysates for cell-free expression from Cube Biotech were successfully used in the following publications:

 ProteinYearAuthor
His-tagged Ups1 together with Mdm35 2019 Miliara X., Tatsuta T., Berry J-L., Rouse S.L.,Solak K., Chorev D.S., Wu D., Robinson C.V., Matthews S. 1
ETB receptor 2018 Rues R-B., Dong F., Dötsch V., & Bernhard F. 2
PRELID3B–TRIAP1 2019 MacVicar T., Ohba Y., Nolte H., Mayer F.-C., Tatsuta T., Sprenger H.-G., Lindner B., Zhao Y., Li J., Bruns C., Krüger M., Habich M., Rlemer J., Schwarzer R., Pasparakis M., Henscke S., Brüning J., Zamboni N., Langer T.2
 
Note that our E.coli lysates require additional components (e.g. amino acids, nucleic acids, DNA polymerase, dialysis devices) that are currently not provided by Cube Biotech. Please refer to the protocols for more details.
In close collaboration with the group of Volker Doetsch and Frank Bernhard, Institute of Biophysics at the University of Frankfurt, Germany, we provide an open system that can be adapted for a range of applications, with dedicated protocols for membrane proteins, continous exchange (dialysis) reactions, and many more. This system takes more work initially than ready to use kits, but provides ultimate flexiblity in reaction variations.
Hi-yield GFP Expression
Fig. 1: Standard vs. HiYield cell-free expression of GFP. 1,7: MW marker, 2-6: HiYield lysate expression; variation of Mg2+ concentration (14, 16, 18, 20,22 mM). 8-12: Standard lysate expression, same variation of Mg2+ concentrations. A Mg2+ optimum was observed at ca. 16 mM for these combinations of cell-free lysates and other reaction components. Yields range from 1.3-5 mg/ml for HiYield reactions, and from 1-3.8 mg/ml for standard reactions. Data kindly provided by Frank Bernhard, University of Frankfurt, Department of Biophysical Chemistry.

 

Features

Purpose Forms nanodisc structures in combination with phospholipids
Specifity Stabilization of membrane proteins
Characteristics MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein
Resulting nanodisc diameter: ca. 7-17 nm
Purity > 90 %
Component Lyophilized MSP protein
Required equipment
 
  • Micropipettor and Micropipetting tips
  • Thermomix / shaking incubator for 500 rpm and 30°C incubation
  • Ultrasonic device (Optional)
  • Dialysis chambers and feeding mix reservoirs, depending on reaction scales. Alternatively home-built dialysis chambers combined with dialysis membrane, cutoff 12-14 kDa. See (1) for details.
Materials
 
  • E.coli lysate, for cell-free protein expression
  • 1,4-Dithiotreitol (DTT)
  • Acetyl phosphate lithium potassium salt (ACP)
  • Acetic Acid
  • Adenosine 5’-triphosphate (ATP)
  • Amino acids for cell-free expression
  • Complete protease inhibitor cocktail
  • Cytidine 5’-triphosphate di-sodium salt (CTP)
  • Dipotassium hydrogen phosphate (K2HPO4)
  • Ethylenediaminetetraacetic acid (EDTA)
  • Folinic acid calcium salt
  • Guanosine 5’-triphosphate di-sodium salt (GTP)
  • 2-(4-(2-Hydroxyethyl)- 1-piperazinyl)-ethane sulfonic acid (HEPES)
  • Magnesium acetate tetrahydrate (Mg(oAc)2)
  • Magnesium chloride hexahydrate (MgCl2)
  • Sodium azide (NaN3)
  • PEG 8,000
  • Phosphoenol pyruvic acid monopotassium salt (PEP)
  • Potassium acetate (KoAc)
  • Potassium chloride (KCl)
  • Potassium dihydrogenphosphate (KH2PO4)
  • Pyruvate kinase (PK)
  • RiboLock RNase inhibitor
  • Sodium chloride
  • Tris base
  • tRNA E.coli MRE 600
  • Uridine 5’-triphosphate tri-sodium salt (UTP)
  • Template DNA
  • Optional: Positive control template DNA
  • Recommended: Suitable affinity chromatography matrix or magnetic beads
  • SDS-PAGE/ Western Blot equipment and antibody
  • Potassium hydroxide (KOH)
  • Sodium hydroxide (NaOH)
 
For membrane protein expression:
  • Pre-assembled nanodisc
  • Detergents, e.g. Decylmaltoside, dodecylmaltoside

Applications

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.
This protocol delineates all necessary steps, chemicals, and solutions to set up a cell-free protein expression reaction in a continuous exchange (dialysis) format. Compared to cell-free reactions in batch format, higher yields can be obtained in dialysis mode, because the reaction is continuously fed through the dialysis membrane, and reaction by-products are removed from the equilibrium. Reaction times can therefore be extended to overnight incubations. Cell-free reactions have vast opportunities because they can be supplemented with isotope labeled amino acids, fluorescence-labeled amino acids or other additives. For membrane protein expression, CECF reactions can be supplemented with detergents, nanodiscs, or other lipid-containing components such as bicelles or liposomes. The novel T7 lysates such as Cellfree E.coli lysates HiYield-T7 already contain the T7 RNA polymerase in optimal quantities. Therefore, this component can be omitted when using these lysates. Cell-free expression should first be optimized in analytical scale (50-100 µL) for parameters such as magnesium concentration, DNA template type and concentration. Optimal conditions can then be linearly scaled up to several mL volume. Please contact us if you have questions or need assistance optimizing a protocol for your application (contact@cube-biotech.com).
   
A.Setup of analytical scale CECF reactions
 
 
  1. Prepare all stock solutions as listed in the PDF-file, set pH with the given amounts of NaOH or KOH, sterile-filter, aliquot, and store.
  2. Prepare a DNA template coding for the protein of interest in a concentration of 0.2-0.5 mg/mL in ddH2O.
  3. Prepare a dialysis reaction container: either assemble a selfbuilt container with a dialysis membrane, or a commercially available reaction container according to the manufacturer’s instructions.
  4. Prepare the required amount of master mix for the number of reactions to be set up.
  5. Reconstitute reaction and feeding mixture in the required amounts.
  6. Check dialysis devices for leakage. Briefly incubate the dialysis membrane in the feeding mix before adding the reaction mix.
  7. Fill reaction and feeding mixture aliquots into reaction containers according to manufacturer’s instructions. When using home-built mini-CECF reactors, ensure that a fresh piece of dialysis membrane is used every time. Always make sure no air bubbles are trapped between the membrane and the solutions.
  8. When using a lysate batch and set of chemical stock solutions for the first time, perform a screen of Mg2+ and K+ concentrations using GFP as positive control. For optimization, screen different amounts of DNA template, and, optionally also concentrations of Mg2+ and K+.
  9. Incubate the CECF reactions overnight at 30°C with gentle shaking or rolling to ensure efficient substance exchange between reaction and feeding mixture.
  10. Spin the mixture at 15,000 rpm for at least 15 min to remove precipitates. Analyze both supernatants and precipitates in SDS-PAGE.
  11. Purify proteins from the reaction mixtures using appropriate affinity chromatography matrices.
  12. . Analyze protein in SDS-PAGE and, optionally, in a Western Blot using an appropriate antibody. For quantification of expressed protein via 35S-Methionine, refer to (1). Measure GFP fluorescence in GFP Assay buffer.

 
   
B.Setup of preparative scale CECF reactions
 
 
  1. After optimizing expression conditions for a given protein in analytical scale, calculate the reaction volume required to obtain the desired amount of recombinant protein.
  2. . Prepare enough DNA template coding for the protein of interest in a concentration of 0.2-0.5 mg/mL in ddH2O.
  3. Prepare a dialysis reaction container: either assemble a selfbuilt container with a dialysis membrane, or a commercially available reaction container according to the manufacturer’s instructions.
  4. Prepare the required amount of master mix for the number of reactions to be set up.
  5. Reconstitute reaction and feeding mixture in the required amounts.
  6. Fill reaction and feeding mixture aliquots into reaction containers according to manufacturer’s instructions.
  7. Incubate the CECF reactions overnight at 30°C with gentle shaking or rolling to ensure efficient substance exchange between reaction and feeding mixture.
  8. Purify proteins from the reaction mixtures using appropriate affinity chromatography matrices.
  9. Analyze protein in SDS-PAGE and, optionally, in a Western Blot using an appropriate antibody. For quantification of expressed protein via 35S-Methionine, refer to (3). Measure GFP fluorescence in GFP Assay buffer

References:

  1. Miliara, Xeni, et al. "Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins." Nature communications 10.1 (2019): 1130.
  2. Rues, Ralf-Bernhardt, et al. "Systematic optimization of cell-free synthesized human endothelin B receptor folding." Methods 147 (2018): 73-83.
  3. Roos, Christian, et al. "High-level Cell-free production of membrane proteins with Nanodiscs." Cell-Free Protein Synthesis. Humana Press, Totowa, NJ, 2014. 109-130.
  4. MacVicar, Thomas, et al. "Lipid signalling drives proteolytic rewiring of mitochondria by YME1L." Nature (2019): 1-5.
Top seller
E.coli cell lysates
No results were found for the filter!
Cell-free E.coli lysate HiYield-T7 Cell-free E.coli lysate HiYield-T7
E.coli lysate for cell-free protein expression. Includes T7 RNA Polymerase. For 1 ml total reaction volume. Requires additional components. Product is shipped on dry ice - additional freight charges apply.   For more information...
Article number: 21031
Sales price: From €124.00 *
Cell-free E.coli lysate Cell-free E.coli lysate
E.coli lysate for cell-free protein expression. For 1 ml total reaction volume. Requires additional components. Product is shipped on dry ice - additional freight charges apply.    
Article number: 21001
Sales price: From €101.00 *
Cell-free E.coli lysate HiYield Cell-free E.coli lysate HiYield
E.coli lysate for cell-free protein expression. For 1 ml total reaction volume. Requires additional components. Product is shipped on dry ice - additional freight charges apply.    
Article number: 21011
Sales price: From €124.00 *
Viewed