Nanodisc membrane scaffold proteins (MSPs)
Membrane scaffold proteins (MSPs) are typically used for the reconstitution of already isolated proteins. A variety of membrane scaffold proteins (MSPs) are available that can be assembled to nanodiscs of different sizes to accommodate proteins with varying numbers of transmembrane domains. His-tagged MSPs allow for easy detection of the nanodisc and even immobilization on SPR chips. On the other hand, if the protein of interest itself carries a His-tag, we recommend to use
untagged MSP proteins. For use in cell-free expression reactions,
assembled nanodiscs are available.
Why Membrane scaffold proteins?
- Variety of lyophilized membrane scaffold proteins available
- Stable, lyophilized protein preparations in convenient aliquots
- Can be combined with different phospholipids
- Detailed protocols and expert support provided
- Also available untagged and as mouse/rat homolog proteins
Nanodisc MSP proteins from Cube Biotech were successfully used in the following publications:
Protein | Year | Author |
Nanolipoprotein particles | 2016 | Wade Z., Kaitlin J., Darryl S., Subhash R., and Marjorie L.1 |
RyR1 and TcdA1 toxin subunit | 2017 | Efremov R., Gatsogiannis C., Raunser S.2 |
unmentioned | 2018 | Carvalho V., Pronk W., Engel, H.3 |
DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a | 2019 | Murugathas T., Zheng H. Y., Colbert D., Kralicek A., Carraher C., Plank N.4 |
BSA | 2019 | Damiati S., Scheberl A., Zayni S., Damiati S., Schuster B., Kompella U.5 |
ETB | 2016 | Rues R.-B., Henrich E., Boland C., Caffrey M., Bernhard F.6 |
TcdA | 2016 | Gatsogiannis C., Merino F., Prumbaum D., Roderer D., Leidreiter F., Meusch D., Raunser S.7 |
Hsc-70 | 2016 | Morozova K., Clement C., Kaushik S., Stiller B., Arias E., Ahmad A., Rauch J., Chatterjee V., Melis C., Scharf B., Gestwicki J., Cuervo A., Zuiderweg E., Santambrogio L.8 |
LacY and XylE (stabilized via MSP1E3D1) | 2019 | Martens C., Shekhar M., Lau A.M., Tajkhorshid E., Politis A.9 |
Features
Purpose | Forms nanodisc structures in combination with phospholipids |
Specifity | Stabilization of membrane proteins |
Characteristics | MSP1D1, MSP1E3D1 or MSP2N2 wild-type protein Resulting nanodisc diameter: ca. 7-17 nm |
Purity | > 90 % |
Component | Lyophilized MSP protein |
Required equipment | - Micropipettor and Micropipetting tips
- Gel filtration column
- FPLC instrument with integrated UV detector and fraction collector
- Magnetic stirrer
- Centrifuge for 2 mL microtubes
- 2 mL microtubes
- Palmitoyl-oleoyl-phosphatidylcholine (POPC)
- Dimyristoyl-glycero-phosphocholine (DMPC) or other suitable phospholipid or phospholipid mixture
- Sodium cholate
- Biobeads SM-2
- Single use syringe
- End-over-end shaker
- Single use needle
- SDS-PAGE reagents and equipment
- Protein concentrator
- 0.45 micron filter
- Optional: Western Blot reagents and equipment and PentaHis Antibody
|
Applications
| |
A. | DMPC: Reconstitution of membrane proteins into nanodisc using His-tagged MSP1D1 protein and DMPC phospholipids |
| - Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 μL of the protein solution at a concentration of 2-5 mg/mL. Note: Depending on the target protein, required protein concentrations might be up to 20 mg/mL.
- Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
- Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
- Calculate the required amount of DMPC to be 80 times the molar quantitiy of the MSP protein (or 1,600 times the molar quantity of the target protein). Example: Target protein has a MW of 45,000 Da. Therefore, 500 μg protein in a 200 μL solution corresponds to 11 nmol of target protein in the 1 ml reaction mix. To obtain a 20 fold excess of MSP protein, use 220 nmol of MSP, i.e. 5.57 mg (MW 25.309 g/mol). For an 80 fold excess of phospholipid, use 17.6 μmol or 11.92 mg of DMPC (MW 677.3 g/mol).
- Resuspend the DMPC in 500 μL ND Lipid Buffer and incubate for 20 min at 37°C to fully solubilize the phospholipid.
- Resuspend the MSP protein in 500 μL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution. Note: The protein was lyophilized from a solution containing 4 mg/ml protein in 20 mM Tris pH 7.4, 100 mM NaCl, 0.5 mM EDTA.
- Add the DMPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
- During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer's instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
- Add the protein/DMPC mix to 750 μL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h
- Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 μL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
- Repeat step 10 at least one more time to ensure complete detergent removal.
- Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
- Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
- Collect fractions of ca. 500 μL size, and analyze the samples by SDS-PAGE. MSP proteins have an apparent molecular mass of around 20 kDa. Note: Optionally, analysis of fractions in the western blot can be done e.g. with Penta His antibodies which recognize the His-tagged MSP1D1 protein.
- Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
- Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.
|
| |
B. | POPC: Reconstitution of membrane proteins into nanodisc using His-tagged MSP1D1 protein and POPC phospholipids |
| - Prepare a membrane protein solution containing your target protein of interest. As a starting point, use 200 μL of the protein solution at a concentration of 2-5 mg/mL. Note: Depending on the target protein, required protein concentrations might be up to 20 mg/mL.
- Determine the molar concentration of target protein solution, using the molecular weight and extinction coefficient e.g. at 280 nm.
- Calculate the required amount of MSP protein to be 20 times the molar quantity of the target protein.
- Calculate the required amount of POPC to be 55 times the molar quantitiy of the MSP protein (or 1,100 times the molar quantity of the target protein). Example: Target protein has a MW of 45,000 Da. Therefore, 500 μg protein in a 200 μL solution corresponds to 11 nmol of target protein in the 1 ml reaction mix. To obtain a 20 fold excess of MSP protein, use 220 nmol of MSP, i.e. 5.57 mg (MW 25.309 g/mol). For a 55 fold excess of phospholipid, use 12.1 μmol or 9.2 mg of POPC (MW 760 g/mol).
- Resuspend the POPC in 500 μL ND Lipid Buffer II and incubate for 20 min at 37°C to fully solubilize the phospholipid.
- Resuspend the MSP protein in 500 μL ND Protein Buffer in a 2 mL microtube. Add the target protein, solubilized in detergent solution. Note: The protein was lyophilized from a solution containing 4 mg/ml protein in 20 mM Tris pH 7.4, 100 mM NaCl, 0.5 mM EDTA.
- Add the POPC solution to the MSP and membrane protein solution, and incubate the entire mix at 4°C for 2 h.
- During the incubation, equilibrate 2.5 g biobeads or similar adsorbant in ND Protein Buffer according to the manufacturer's instructions. Degas the solution by ultrasound to remove any oxygen solubilized in the biobead solution.
- Add the protein/POPC mix to 750 μL of the biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
- Spin the solution at 10-12,000 x g for 2 min, and transfer the supernatant to a fresh tube. Add 750 μL of fresh equilibrated biobead solution and incubate at 4°C on an end over end shaker for 8-12 h.
- Repeat step 10 at least one more time to ensure complete detergent removal.
- Remove the supernatant, and filter the nanodisc mix through a 0.45 micron filter to remove precipitates that might have formed during incubation.
- Apply the nanodisc mix on a gel filtration column. Apply the mix in several portions if necessary. Monitor absorbance at 280 nm.
- Collect fractions of ca. 500 μL size, and analyze the samples by SDS-PAGE. MSP proteins have an apparent molecular mass of around 20 kDa. Note: Optionally, analysis of fractions in the western blot can be done e.g. with Penta His antibodies which recognize the His-tagged MSP1D1 protein.
- Concentrate the elution fractions which contain the nanodiscs with inserted membrane protein using protein concentrators.
- Freeze the nanodiscs containing membrane proteins at -80°C in a solution containing 10% glycerol for future use, or use them directly in the desired experiment.
|
References
- Zeno, Wade F., et al. "Dynamics of crowding-induced mixing in phase separated lipid bilayers." The Journal of Physical Chemistry B 120.43 (2016): 11180-11190.
- Efremov, Rouslan G., Christos Gatsogiannis, and Stefan Raunser. "Lipid nanodiscs as a tool for high-resolution structure determination of membrane proteins by single-particle cryo-EM." Methods in enzymology. Vol. 594. Academic Press, 2017. 1-30.
- Carvalho, Vanessa, Joachim W. Pronk, and Andreas H. Engel. "Characterization of Membrane Proteins Using Cryo‐Electron Microscopy." Current protocols in protein science 94.1 (2018): e72.
- Murugathas, Thanihaichelvan, et al. "Biosensing with Insect Odorant Receptor Nanodiscs and Carbon Nanotube Field-Effect Transistors." ACS applied materials & interfaces 11.9 (2019): 9530-9538.
- Damiati, Samar, et al. "Albumin-bound nanodiscs as delivery vehicle candidates: Development and characterization." Biophysical chemistry 251 (2019): 106178.
- Rues, Ralf-Bernhardt, et al. "Cell-free production of membrane proteins in Escherichia coli lysates for functional and structural studies." Heterologous Expression of Membrane Proteins. Humana Press, New York, NY, 2016. 1-21.
- Gatsogiannis, Christos, et al. "Membrane insertion of a Tc toxin in near-atomic detail." Nature structural & molecular biology 23.10 (2016): 884.
- Morozova, Kateryna, et al. "Structural and biological interaction of hsc-70 protein with phosphatidylserine in endosomal microautophagy." Journal of Biological Chemistry 291.35 (2016): 18096-18106.
- Martens, Chloe et al. "Integrating hydrogen–deuterium exchange mass spectrometry with molecular dynamics simulations to probe lipid-modulated conformational changes in membrane proteins" Nature Protocols (2019): 18096-18106.