Cell-free E.coli lysate

Order number: 21001

€142.00*

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Description

Our E.coli cell-free lysates are the isolated protein purification machinery of bacterial cells. They create a high-yielding and adjustable cell-free protein expression system. The advantages of cell-free protein expression are numerous, among other things it enables a scientist to:
  • For direct stabilization and co-translational integration of membrane proteins into nanodiscs
  • Expression of otherwise toxic proteins
There are three cell-free lysates available:
ProductArticle NumberAverage YieldsVolume per vial
Cell-free E.coli lysate210011.3-5 mg/ml350 µl
Cell-free E.coli lysate HighYield21011>1.3-5 mg/ml350 µl
Cell-free E.coli lysate HighYield-T721001up to 6 mg/ml330 µl
Additionally required equipment & materials
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.
Additional materials & chemicals
  • 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)
  • PEG 8,000
  • Phosphoenol pyruvic acid monopotassium salt (PEP)
  • Potassium acetate (KoAc)
  • Potassium chloride (KCl)
  • Potassium dihydrogenphosphate (KH2PO4)
  • Potassium hydroxide (KOH)
  • Pyruvate kinase (PK)
  • RiboLock RNase inhibitor
  • Sodium azide (NaN3)
  • Sodium chloride
  • Tris base
  • tRNA E.coli MRE 600
  • Uridine 5’-triphosphate tri-sodium salt (UTP)
Project specific materials

Citations

TitleYearAuthor
High-Level Cell-Free Production of Membrane Proteins with Nanodiscs 2013 Roos C., Kai L., Haberstock S., Proverbio D., Ghoshdastider U., Ma Y., Filipek S., Wang X., Dötsch V., Bernhard F.
Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins 2019 Miliara X., Tatsuta T., Berry J-L., Rouse S.L.,Solak K., Chorev D.S., Wu D., Robinson C.V., Matthews S.
Systematic optimization of cell-free synthesized human endothelin B receptor folding 2019 Rues R-B., Dong F., Dötsch V., & Bernhard F.
Systematic optimization of cell-free synthesized human endothelin B receptor folding 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.
Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy 2022 Poláchová E., Back K., Heuten E., Stanchev S., Tichá A., Lampe P., Majer P., Langer T., Lemberg M.K., Stříšovský K.

Lab Results

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.
Performance of Cube Biotech Cell-free expression lysated HighYield
Figure 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.