Nanodisc MSP proteins

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
-	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

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

All protocols are also avaible as PDF-Files on the Protocols & Datasheets page.


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.

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.

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.