Editing Membrane Protein Production

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2253291/

Spheroplasts of E. coli and a cytoplasmic membrane fraction were prepared according to the protocols by Weiss (1976) and Tai and Kaplan (1985) with the following modifications: E. coli cells were collected by centrifugation, washed twice with 0.1 M Tris-HCl (pH 8.0) buffer, and resuspended in 0.1 M Tris-HCl (pH 8.0) buffer containing 20% (w/v) sucrose, to adjust an optical density of the cell suspension to 10 at 600 nm. A cocktail of protease inhibitors (Roche) was added at this stage. The temperature was adjusted to 37°C, and a solution of lysozyme (2 mg/mL) was added slowly, under constant stirring, until the final concentration of lysozyme of 0.1 mg/mL was reached. The cell suspension was incubated at 37°C for another 15 min with stirring. A solution of 0.1 M EDTA (pH 7.0) was added slowly under continuous stirring until the final concentration of EDTA of 10 mM was reached. The incubation continued for another 10 min.



Spheroplasts were centrifuged at 12,000g for 20 min. The pellet was collected and washed once with 0.1 M Tris-HCl (pH 8.0) containing 20% sucrose. Spheroplasts were centrifuged again at 12,000g for 20 min. The pellet of spheroplasts was resuspended in ice-cold water, which resulted in osmotic lysis. The remaining intact cells were broken up by mild sonication. Immediately, 1 M Tris buffer (pH 8.0) containing protease inhibitors was added to produce a final Tris concentration of 50 mM. A 1 M solution of MgCl2 was added to achieve a final concentration of 1 mM. A solution of DNAse I was added, the cell-free extract was briefly sonicated, and further incubated on ice for 1 h. The extract was centrifuged at 150,000g for 1 h, and the membrane pellet was washed with Tris buffer and centrifuged again at 150,000g for 1 h. The membrane pellet was resuspended in a small volume of Tris buffer, frozen in liquid nitrogen, and stored at −80°C.



https://www.nature.com/articles/s41598-018-19749-5/

Membranes from E. coli cells were prepared using previously published protocols 34. Briefly, the E. coli cell pellet (2 g) was washed twice with 0.1 M Tris-HCl, pH 8.0 (20 ml) and resuspended in cold 0.1 M Tris-HCl containing 20% sucrose (30 ml). To the cell suspension, Halt protease inhibitor cocktail (Fisher Scientific, Pittsburgh, PA) was added (final concentration of 10 µl/ml), incubated at 37 °C for 15 min, followed by lysozyme treatment (final concentration of 0.1 mg/ml) and incubation for 15 min at 37 °C with mild agitation. To this mixture, ethylenediaminetetraacetic acid (EDTA) was added (final concentration of 10 mM) and incubated further for 10 min at 37 °C with mild agitation. The mixture was centrifuged at 12000 g for 20 min and washed with 0.1 M Tris-HCl containing 20% sucrose (20 ml). The pellet was then resuspended in ice-cold water (2 ml) and briefly sonicated. To this suspension, Halt protease inhibitor cocktail (final concentration of 10 µl/ml), DNAse I (1000 U), MgCl2 (final concentration of 1 mM) and Tris-HCl (final concentration of 50 mM) were added, briefly sonicated and incubated on ice for 1 h. This suspension was centrifuged at 100,000 g for 1 h and the membrane pellet was washed with 50 mM Tris-HCl (20 ml) and centrifuged again at 100,000 g for 1 h. The membrane pellet was resuspended in 10 ml of 50 mM Tris-HCl and stored at −80 °C in aliquots. Membrane protein was quantified with a Bradford dye-binding method (Bio-Rad Laboratories).



The cell-free MembraneMax Protein Expression Kit (Invitrogen, Carlsbad, CA) and the pET26shΔCB1T4Lhis6 or pET26FlaghΔCB1T4Lhis6 plasmid DNA were used for expression of CB1 receptor variants according to the manufacturer’s recommended protocol with minor modifications. The in vitro expression reaction, containing plasmid DNA (1 μg), and Porcine Optizyme RNase Inhibitor (400 units) (Fisher Scientific, Pittsburgh, PA) in reaction buffer mixture (final volume of 100 μl), was mixed at 300 rpm on a plate shaker for 30 min at 30 °C. Then 100 μl of feed buffer mixture was added (final reaction volume of 200 μl) and the incubation continued for another 90 min according to the manufacturer’s recommendation. The expression of hΔCB1T4Lhis6 protein was confirmed by SDS-PAGE analysis in AnykD Mini-PROTEAN® TGX™ precast polyacrylamide gels (Bio-Rad), followed by western blot analysis using his-tag based immuno-detection as detailed below. The total protein concentration was determined using Quick Start™ Bradford Protein Assay kit (Bio-Rad) according to the manufacturer’s recommended protocol and the absorbance values were measured at 595 nm using the EnVision™ Multilabel Plate Reader (Perkin Elmer, Waltham MA).