Cell-free E.coli lysate

Order number: 21001

€142.00*

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Lysate Type

µL

Quantity

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:

Product	Article Number	Average Yields	Volume per vial
Cell-free E.coli lysate	21001	1.3-5 mg/ml	350 µl
Cell-free E.coli lysate HighYield	21011	>1.3-5 mg/ml	350 µl
Cell-free E.coli lysate HighYield-T7	21031	up to 6 mg/ml	330 µl

Datasheets

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	Template DNA Optional: Positive control template DNA Recommended: Suitable affinity chromatography matrix or magnetic beads SDS-PAGE/ Western Blot equipment and antibody For membrane protein expressions: Pre-assembled MSP nanodisc For membrane protein expressions: Detergents, e.g. Decylmaltoside, dodecylmaltoside

Citations

Title	Year	Author
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.