The overall goal of this procedure is to incorporate an A, b, C transporter. In this case, the maltose transporter malf GK two into soluble membrane nano disk particles. This is accomplished by first mixing the membrane scaffold protein MSP, with the transporter and the correct amount of lipids in detergent mycells.
The second step is to add polystyrene beads and to gently rock the mixture overnight, the detergent is progressively absorbed onto the beads, allowing the formation of the nano disk particles. Next, the beads are removed by sedimentation. The final step is to separate the properly formed discs from aggregates and unassembled scaffold proteins by gel filtration chromatography.
Finally, the quality of the discs is assessed by native gel electrophoresis or light scattering spectroscopy. The main advantage of this technique over other methods like liposomes, is that membrane proteins and discs are perfectly soluble in aqueous solution. In addition, the protein domains are now accessible from both side.
Since now is no more membrane compartment Using membrane scaffold proteins of different lengths, I was able to trap and study the different ligament states of membrane proteins. Most importantly, this method can help answer key questions in the membrane proteomic field, such as the identification of novel binding partners of a, b, C transporters. In fact, the implications of this technique extend further toward therapy because it can help the discovery of activators or inhibitors of membrane proteins and receptors.
Though this method seems simple, caution should be taken when choosing the correct protein lipids ratio. Generally, individuals new to this method while struggle in calculating the correct molder ratio or doing the correct control that ensure the quality of the preparation. Therefore, a visual demonstration of this method is very useful for a new student to learn the various steps and potential difficulties.
To begin gro hexa histidine tagged MSP in e coli strain BL 21 DE three from the plasmid P MSSP 1D one induced protein production when the measured optical density at 600 nanometers is about 0.5 for three hours at 37 degrees Celsius, harvest the cells by centrifugation at 5, 000 Gs for 10 minutes at four degrees Celsius. Re suspend the pellet in TSG 10 buffer containing 100 micromolar PMSF lice the cells with a French press three times at 8, 000 PS.I remove the insoluble material by centrifugation at 5, 000 Gs for 10 minutes at four degrees Celsius. Then isolate the soluble fraction containing the MSP by ultracentrifugation at 125, 000 Gs for 45 minutes.
Purify the MSP by nickel chelating chromatography using approximately 1.5 milliliters of nickel seros high performance in TSG 10 buffer. Wash away the contaminants with T SG 10 buffer containing 50 millimolar ILE elute. The MSP with TSG 10 buffer containing 600 millimolar I midaz after dialyzing.
The purified protein in TSG 10 buffer MSP can be stored at minus 70 degrees Celsius and a protein concentration of approximately 10 to 15 milligrams per milliliter. The MALF GK two complex, his tagged at the C terminus of MAL K, is expressed from plasmid. PED 22 FG K grown in e coli strain BL 21 DE three induce protein production when the measured optical density at 600 nanometers is about 0.5 with 0.2%L Arabs for three hours at 37 degrees Celsius following cell lysis and centrifugation.
As before, we suspend the membrane pellet in TSG 20 buffer to a final concentration of five milligrams per milliliter. Next, solubilize the material with 1%weight per volume GDM for three hours at four degrees Celsius with gentle shaking. Remove the insoluble material by ultracentrifugation as for purification of MAL FTK two.
Then collect the supernatant containing the solubilized mal FTK two complex and purify again with nickel chelating chromatography. Further purify the complex by gel filtration chromatography in TSGD buffer on a SUEx 200, HR ten three hundred column at a flow rate of 0.5 milliliters per minute. To prepare phospholipids, an e coli total lipid extract dissolved in chloroform is separated into 1000 anomal aliquots in screw cap microfiche tubes evaporate the solvent under a gentle stream of nitrogen and drive further overnight in a vacuum.
Desiccate next, dissolve the lipid film in TS buffer at five NMO final concentration and vortex vigorously then pulse sonicate five times for approximately five seconds. The dissolved lipids will appear slightly opaque due to their general insolubility in aqueous TS buffer, but they should remain in suspension. Add DDM to a final concentration of 0.5%At this point, the solution will become clear place lipid mixture in a water bath and puls sonicate five times for approximately five seconds.
The lipid mixture can be stored at four degrees Celsius for a maximum of one week. To prepare the bio beads place approximately 10 to 15 milliliters of bio beads into a 50 milliliter tube. First, wash the beads with 50 milliliters of 100%methanol.
Allow the beads to settle by gravity and then gently pour off the solution. Repeat the process once for methanol and then twice each for 95%Ethanol milli Q water and finally TS buffer. Begin nano discrete constitution by diluting MSP in TSGD buffer to the final concentration of approximately seven milligrams per milliliter or 0.3 millimolar.
To ensure a successful experiment, it's important to choose the correct ratios of lipids and proteins to be mixed together. Mix approximately two M of purified malf GK two complex at a protein to MS P to lipid ratio of one to three to 60 in TSGD buffer for a final volume of 300 microliters here, a sample of excess lipid in the ratio of one to three to 400 is also prepared as a negative control at a 50 microliters of bio bead suspension to the tube and incubate the mixture overnight on a rocking table at four degrees Celsius the following day, sediment the beads by gravity. Once the beads have settled pipe at the solution through a narrow tip to avoid as much bio beads as possible.
Remove large precipitates by ultracentrifugation at 100, 000 GS for 20 minutes. Proceed to purify the discs by gel filtration chromatography as performed earlier in TSG 10 buffer. Next, inject an Eloqua on the same gel filtration column to test the stability of the preparation.
Then pool the fractions containing the discs. The quality of the preparation is assessed by native gel electrophoresis. Analyze one micromolar of nano discs to assess the quality of the reconstitution.
Also assess the binding of mal E to the mal ftk two complex reconstituted in nano discs. After electrophoresis, stain the gel with kumasi blue for 10 minutes and detain for approximately one hour. To perform dynamic light scattering or DLS first purify nano discs by gel filtration chromatography as before, using a SUEx 200 HR ten three hundred column at a flow rate of 0.1 milliliters per minute.
Pool fractions containing nano discs, and concentrate to approximately 10 milligrams per milliliter with an AmCon centrifugal filter. Then filter the sample twice. Perform analysis by DLS using a dynapro nano star instrument in a one microliter inner volume quartz vete fit data.
Using the dynamic software to estimate the diameter and molecular weight of the particles, determine the A TPAs activity of mal FTK two reconstituted nano discs using a chole metric assay. First mix one micromolar of purified discs and one millimolar a TP with increasing amounts of mal e. Incubate the samples at 37 degrees Celsius for 20 minutes.
Then measure the release of inorganic phosphate at 660 nanometers. Compare the amount of phosphate released to a standard curve generated with a phosphorus standard solution. The nano discs are purified by gel filtration chromatography.
The chromatogram shows that the majority of the reconstituted discs elute as a single peak, whereas discs made with excess lipids elute in the void volume, and as a series of broad peaks, the quality of the disc preparation is further analyzed by native gel electrophoresis discs reconstituted with a limited amount of lipids migrate as a sharp band on the native gel, whereas those reconstituted in the presence of an excess of lipids migrate as smears. Analysis by DLS shows that the disc population is homogeneous with an average radius of 5.0 nanometers. Based on the DLS approximation, the reconstituted discs have an apparent molecular weight of 215 kilodaltons samples reconstituted in the presence of excess lipids display widely distributed radii around 100 nanometers, which is typical for non homogeneous samples.
The activity of the MAL FTK two complex is assessed by measuring the rate of a TP hydrolysis and the affinity for interacting partners such as the maltose binding protein, MAL E, the maltose binding protein. MAL E binds with a high affinity to the MAL FTK two transporter using non denaturing gel electrophoresis. It is possible to detect a complex between MAL E and MAL ftk two, the stimulation of the MAL KA TPA activity by MAL E is shown here as a function of MAL e concentration.
The making of the nano disc is relatively simple, yet small deviations from optimal conditions can lead to irreversible protein aggregation once mastered, this technique can be done in less than two days. While attempting this procedure, it's important to remember the importance of choosing a correct protein lipid ratio because an excess amount of lipids will lead to production of large poly disperse liposome like particles. It is this crucial to carefully analyze the quality of the reconstituted material using techniques other than gel filtration, such as light scattering spectroscopy, analytical ultracentrifugation, or more simply native gel electrophoresis.
Remember, the type of phospholipid incorporating the disc as well as the of membrane scaffold protein can contribute to the efficiency of the reconstitution. After watching this video, we hope you got a good understanding of how to generate soluble and homogenous nano discs containing your membrane protein of choice. Good luck with your experiments.
