E coli polar lipid extract

Manufactured by Avanti Polar Lipids

Sourced in United States

E. coli polar lipid extract is a laboratory product derived from the cell membranes of Escherichia coli bacteria. It contains a complex mixture of polar lipids, including phospholipids and glycolipids, which are the primary structural components of bacterial cell membranes. This extract is commonly used in research applications involving the study of membrane structure, function, and lipid-protein interactions.

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45 protocols using e coli polar lipid extract

Preparation of Bacterial Lipid Stocks

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To prepare the lipid stock solution, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen and stored at −80°C. To prepare a 2 mg/mL aqueous suspension stock of lipopolysaccharide, LPS from E. coli EH100 (Ra mutant; Sigma) was dissolved in water and sonicated for 30 min. The lipopolysaccharide stock solution was flash frozen in liquid nitrogen and stored in aliquots at −80°C.
To prepare the fluorescent lipid stock solutions for flow cytometry, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in chloroform with 1% (molar ratio) ATTO-488 DPPE, ATTO-565 DPPE, or ATTO-647N DPPE (ATTO-TEC GmbH). Lipid mixtures were then dried into a film, hydrated, and lyophilized overnight. The lipid mixtures were then dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen, and stored at −80°C.

Falchi F.A., Taylor R.J., Rowe S.J., Moura E.C., Baeta T., Laguri C., Simorre J.P., Kahne D.E., Polissi A, & Sperandeo P. (2022). Suppressor Mutations in LptF Bypass Essentiality of LptC by Forming a Six-Protein Transenvelope Bridge That Efficiently Transports Lipopolysaccharide. mBio, 14(1), e02202-22.

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Reconstitution of BAM Complex in E. coli Lipids

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E. coli polar lipid extract, purchased as powder from Avanti Polar Lipids (Alabaster, AL), was dissolved in 80:20 (v/v) chloroform/methanol at 20 mg/mL. Appropriate volumes were dried to thin films in clean Pyrex tubes at 42 °C under N₂ gas, and were further dried by vacuum desiccation for at least 3 h. The BAM complex in TBS pH 8.0, 0.05% (w/v) DDM was mixed with E. coli polar lipid extract films solubilised in TBS pH 8.0, 0.05% (w/v) DDM in a 1:2 (w/w) ratio. Empty liposomes were prepared by mixing lipid with an equivalent volume of buffer. To remove detergent and promote liposome formation, the mixtures were dialysed against 2 L of 20 mM Tris–HCl pH 8.0, 150 mM KCl using 12–14 kDa MWCO D-Tube™ Maxi Dialyzers (Merck) at room temperature for 48 h with a total of four buffer changes. Following dialysis, the proteoliposomes were pelleted twice by ultracentrifugation at 100,000 × g for 30 min at 4 °C and were resuspended in TBS pH 8.0. Protein concentration was determined using a BCA assay (Thermo Scientific) and successful reconstitution was confirmed by SDS–PAGE.

Schiffrin B., Machin J.M., Karamanos T.K., Zhuravleva A., Brockwell D.J., Radford S.E, & Calabrese A.N. (2022). Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding. Communications Biology, 5, 560.

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SecYEG Reconstitution in Lipid Vesicles

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SecYEG complexes were reconstituted into lipid vesicles and planar bilayers as previously described.^{17 (link)} In brief, to 20 mg ml⁻¹ of E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL) we sequentially added 50 mM K-HEPES pH 7.5, 6% deoxy Big-CHAP (Affymetrix Anatrace, Maumee, OH, USA) and SecYEG in detergent (protein to lipid ratio of 1 : 50) at room temperature. For LRET experiments a DOPE : DOPG (Avanti Polar Lipids, Alabaster, AL) mixture (mass ratio 7 : 3) was used instead of E.coli polar lipid extract due to unfavorable unspecific binding of Tb³⁺ to this lipid extract. Subsequent to detergent removal by Bio-beads SM2 (Bio Rad), the proteoliposomes were harvested by ultracentrifugation (80 min at 100 000×g) and resuspended at a lipid concentration of 5–10 mg ml⁻¹ in a buffer containing 50 mM HEPES (pH 7.0), 10% glycerol and protease inhibitor. To show channel functionality we tested reconstituted SecYEG mutants for their translocation capability as previously described.^{24,25 (link)} In the presence of both ATP and SecA proOmpA-DHFR is translocated into proteo-LUVs and is therefore not accessible to a subsequent proteinase K digest (Fig. S1†).

Winkler K., Karner A., Horner A., Hannesschlaeger C., Knyazev D., Siligan C., Zimmermann M., Kuttner R., Pohl P, & Preiner J. (2020). Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP. Nanoscale Advances, 2(8), 3431-3443.

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Asymmetric pH Membrane Reconstitution

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Reconstitutions into the asymmetric pH membranes was accomplished through n-decyl-β-D-maltoside (DM, Anatrace) detergent by reconstituting the protein at a given pH and pelleting of the vesicles by ultracentrifugation followed by a gentle resuspension to the desired pH on the external side of the liposomes. Wild-type EmrE was reconstituted in DMPC or E. coli polar lipid extract (Avanti Polar Lipids, Inc.) where the pH values inside/outside the vesicles were set as pHⁱⁿ/pH^out: 6.2/6.2, 6.2/8.7, 8.7/6.2, and 8.7/8.7. The buffer condition was 50 mM sodium phosphate and 20 mM NaCl. After reconstitution the proteoliposomes were centrifuged for 1.5 hr at 130,000 ×g and resuspended in the same buffer as that in the interior of the vesicles. This cycle was done a total of two times to ensure control of the inside pH. Prior to collection of the fluorescence spectra, the proteoliposomes were diluted 30-fold with the external buffer. After collection of the ΔpH experiments, a small amount of stock DDM was added (final ratio of DDM/lipid = 1/50, mol/mol) to disturb the pH gradient. Since the volume outside the vesicles was greater than that inside, the pH of the interior changed to the external value.

Gayen A., Leninger M, & Traaseth N.J. (2016). Protonation of a Glutamate Residue Modulates the Dynamics of the Drug Transporter EmrE. Nature chemical biology, 12(3), 141-145.

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Bacterial Membrane Lipid Interactions

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E. coli polar lipid extract (Avanti Polar Lipids) was dried in a rotational evaporator and lyophilized overnight. Lipids were resuspended in buffer A at 20 mg/ml and stored at -80°C. B. subtilis lipids / membranes were isolated as described above. For liposome preparation, E. coli lipids were diluted to 5 mg ml^-1 in buffer A. Either E. coli lipids or isolated B. subtilis membranes were extruded 20 times through a 400 nm pore size membrane (Millipore). 1 ml of liposomes were incubated for 1h at RT with 100 µl of PHB domain solution (0.8 mg ml^-1) rolling with 0.5 revolution per second. Protein concentration was determined using BCA assay (Pierce). As a control the PHB domain was incubated with buffer only and used for all steps mentioned below. The mixture was centrifuged at 200,000xg for 20 minutes at 4°C. The pellet was resuspended in an equal volume and centrifuged again. Finally the pellet was again resuspended in an equal volume. All samples were analysed by SDS-PAGE. Sedimentation of DynA was performed identical to PHB domain. DynA was purified according to Bürmann et al, 2011 [36 (link)].

Bach J.N, & Bramkamp M. (2015). Dissecting the Molecular Properties of Prokaryotic Flotillins. PLoS ONE, 10(1), e0116750.

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Proteoliposome Preparation and Characterization

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Proteoliposomes were created as previously described with modifications [34 (link)]. First, 3 mg of E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, Alabama, USA) were suspended in 1 mL 100 mM phosphate buffer (pH 7.5) and extruded 19 times through a polycarbonate membrane (Whatman^®, Nucleopore™ PC Track-Etched Membrane, 19 mm, 0.4 μm) using a mini extruder (Avanti Polar Lipids). The liposomes were titrated with Triton X-100 until saturation and 50 µmol of the WalK kinase as well as different ratios of YycH and YycI were added to the reaction. Triton X-100 was subsequently removed from the liposomes using an adsorbent (Bio-Beads SM-2, Bio-Rad). The liposomes were ultracentrifuged, resuspended in 1 mL phosphorylation buffer (50 mM HEPES, 500 mM KCl, 5 mM MgCl₂, 0.5 mM dithiothreitol, 3.5% (v/v) glycerol, pH 8), and extruded again as described above. After another ultracentrifugation step, the liposomes were resuspended in 100 µL of phosphorylation buffer and inorganic phosphate content of every batch of liposomes was quantified as previously described [30 (link)]. The proteoliposomes were used in phosphorylation assays or stored at 4 °C.

Gajdiss M., Monk I.R., Bertsche U., Kienemund J., Funk T., Dietrich A., Hort M., Sib E., Stinear T.P, & Bierbaum G. (2020). YycH and YycI Regulate Expression of Staphylococcus aureus Autolysins by Activation of WalRK Phosphorylation. Microorganisms, 8(6), 870.

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Purification and Characterization of Membrane Proteins

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Isopropyl-β-d-thiogalactopyranoside (IPTG), Tris(2-carboxyethyl)phosphine (TCEP), n-dodecyl-β-d-maltopyranoside (DDM) and precast SDS–PAGE gels were from Generon. All aromatic acids, l-arabinose, cholesteryl hemisuccinate Tris salt (CHS), 0.4–0.6 mm acid-washed glass beads, valinomycin and Proteinase K-agarose were from Sigma–Aldrich. HisTrap columns, PD-10 and PD SpinTrap G-25 gel filtration columns, Superdex 200 10/300 GL column, size exclusion column standards, and nitrocellulose membrane were from GE Healthcare. Chemiluminescence reagents (LumiGLO) were from Cell Signaling Technologies. E. coli strain BL21-AI, LDS loading buffer, V5-HRP, pyranine and gels and reagents for blue native PAGE were from Life Technologies. Centrifugal concentrators were from Millipore. The detergent compatible Lowry assay was purchased in kit format from ThermoFisher. E. coli polar lipid extract and egg phosphatidylcholine were from Avanti Polar Lipids.

Pernstich C., Senior L., MacInnes K.A., Forsaith M, & Curnow P. (2014). Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1. Protein Expression and Purification, 101, 68-75.

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Protein Reconstitution in Proteoliposomes

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C-terminally His-tagged proteins were overexpressed in E. coli BL21(DE3) cells and purified using immobilized metal affinity chromatography as previously described (2 (link)). Reconstitution of the purified proteins into proteoliposomes was performed using E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL) at a lipid-to-protein ratio of 5, essentially as previously described (34 (link)).

Călinescu O., Dwivedi M., Patiño-Ruiz M., Padan E, & Fendler K. (2017). Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter. The Journal of Biological Chemistry, 292(19), 7932-7941.

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Reconstitution of SecYEG in E. coli Lipids

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SecYEG was reconstituted into E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL, USA) vesicles pre-dissolved in deoxy-BigChap (Anatrace, Maumee, OH, USA) as previously described [3 (link)]. Biobeads SM2 (Biorad, Hercules, CA, USA) were added to remove the excess detergent and the resulting turbid suspension was pelleted at 100.000 g. The resulting pellet was resuspended and extruded through a 100 nm filter. Mass ratios of protein to lipid of 1:54 to 1:108 were used.

Knyazev D.G., Kuttner R., Bondar A.N., Zimmerman M., Siligan C, & Pohl P. (2020). Voltage Sensing in Bacterial Protein Translocation. Biomolecules, 10(1), 78.

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Reconstitution of Cgs Protein in Nanodiscs

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E. coli polar lipid extract (Avanti Polar Lipids) was solubilized in chloroform, dried under nitrogen gas to form a lipid film, and stored under vacuum overnight. The lipid film was resuspended at a concentration of 25 mM in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 300 mM sodium cholate. Purified Cgs, the MSP1D1 membrane scaffold protein¹³ and lipids were mixed at a molar ratio of 1:4:100 in buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl and incubated for 30 min. at 4 °C. Detergents were removed by incubation with 100 mg Bio-Beads SM2 (Bio-Rad) overnight at 4 °C. The Cgs-nanodisc complexes were purified using a Superose 6 Increase 10/300 column (GE Healthcare) in a buffer containing 25 mM HEPES, pH 7.5, and 150 mM NaCl.

Sedzicki J., Ni D., Lehmann F., Stahlberg H, & Dehio C. (2024). Structure-function analysis of the cyclic β-1,2-glucan synthase from Agrobacterium tumefaciens. Nature Communications, 15, 1844.

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