SMALP Protocols

SMALP network

fly

SMALP Protocols

The following protocols are being standardized based on comparative studies of over 50 membrane proteins which have been purified intact from Escherichia coli, Pichia pastoris, insect, mammalian cells, and brain, fly and plant tissue using the SMALP system:

Production of activated styrene maleic acid
Optimization for pH for SMALPing membranes
DIBMA protocol, Sandro Keller's talk, Cube Biotech DIBMA protocol, services and review
SMALPing ABC transporters, BK channels, dopamine and SMO GPCRs from insect and mammalian cell lines
Solubilization of raft vs nonraft lipids from T cells
Screening polymers for MP solubilization & purification
SMA-PAGE and native gel electrophoresis
Flow cytometry, antigens and antibody studies with SMALPs
Proteomic and lipid analysis of the SMALP'd metabolon
Mass spectometry analysis of lipids and hENT1 and GlpG in SMALPs
SMALP complexes by XRD and EM; AcrB and ACIII structures.
Biophysical analysis of SMALPs by methods including:

Protocols for using the SMALP system are described in papers including

A Method for Detergent-free isolation of Membrane Protein with its Local Lipid Environment (Nat Protoc.)
Detergent-Free Membrane Protein Purification Using SMA Polymer (MIMB, 2022)

The discovery of SMALPs was first reported in 2009. Reviews on the progress since include:

Overduin M, Trieber C, Prosser RS, Picard LP, Sheff JG. Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids. Membranes 11(6):451.
Sun C, Gennis RB. Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs. Chem Phys Lipids. 221:114-119
Brown CJ, Trieber C, Overduin M, Structural biology of endogenous membrane protein assemblies in native nanodiscs, Curr Opin Struct Biol 69, 70-77
Ravula T, Hardin NZ, Ramamoorthy A. Polymer nanodiscs: advantages and limitations. Chem Phys Lipids 219:45-49.

Join us for the next meeting which includes talks from a diverse array of experts and a practical session on SMALP protocols and R&D priorities for the next year.

Check out this video on the SMALP method by CUBE Biotech:

Here are talks about SMALPs and membrane proteins by Alice Rothnie (Aston U) and Steve Harborne (Peak Proteins) and hosted by Nature Research.

Feel free to discuss below or post questions on Linkedin in or tweet @smalpnet.

Receive nanodisc protocol tips

You can unsubscribe anytime.

Meeting Questions & Answers

Extraction of membrane proteins

Q: Does SMA generally give low recovery of protein after extraction (with reference to GPCRs)?
A: It depends. For some proteins it out performs detergents, for others it is worse. Consider trying a variety of polymers to improve yields of solubilization, extraction and activity, then try using different tags at different positions.

Q: It is difficult to extract proteins from Pichia pastoris
A: Preparing spheroplasts prior to solubilisation may help.
A2: Adding DMPC to membrane samples can aid solubilisation (e.g. often used for rhodopsin extraction due to scarcity of native lipids in membranes with densely packed protein). If overexpression of a target protein is high, adding DMPC may help with solubilisation efficiency.

Q: When solubilising insect/HEK cells a white film is often observed at the top of the tube after solubilisation and a high-speed centrifugation step.
A: Could be cholesterol.

Q: Can polymers be mixed to optimise extraction?
A: In theory yes, but only those with the same charge or they would likely precipitate. Fluorescent polymers could be used to find out whether different polymers co-SMALP.

Working with SMALP in plasticware

Q: What spin concentrators work for SMALPs?
A: Some people recommended PALL brand on performance and cost.

Q: What tubes are good for storing SMALP samples?
A: If concerned about lipid/polymer sticking to tubes try those designed for lipidomics which minimise hydrophobic interactions.

Q: Are there any ideas for best practice with using concentrators?
A1: Use a PEG 2000/3000 solution wash prior to use to prime concentrators
A2: A 10 kDa concentrator won’t let through polymer of 10 kDa due it extended size. For good concentrator performance you need to go up sizes, which is protein dependent and needs to be optimised.
A3: A 100 kDa cut off concentrator will retain most protein-SMALPs and let polymer through, especially for free polymer removal. This may also prevent aggregation.
A4: Another approach is to use a 50 kDa dialysis membrane to remove free polymer prior to binding to an affinity resin
A5: Always use cooled centrifuges and do not spin too fast (this also applies for detergents)
A6: Always mix what’s in concentrators every 2-5 mins to prevent locally high concentrations which may drive aggregation in proteins
A7: Try the upside down concentrators, e.g. from Generon.

Resins and tags

Q: What resins work well with protein-SMALPs? Use NTA or other base resin?
A1: For His tags the resin brand makes a difference in some people’s experience. HisPur seems to work best for SMALP proteins. Pierce Cobalt resin (Co2+ version of the same resin) was also recommended. Consensus what that it is another thing that just needs optimisation!
A2: Magnetic resins can work well. Concentration-dependent aggregation has been observed in very small batch sizes/amounts of resin.
A3: Binding to Ni-NTA is often less efficient in protein-SMALPs so overnight binding in loose resin is encouraged!
A4: Use fresh metal affinity resins or columns to purify His-tagged proteins in SMALPs as they will provide better separation than re-used resins/columns.

Q: What is the recommended length of His tag for successful purification?
A1: In eukaryotic cells, spreading out the His along the tag can work better but this is difficult when using commercial antibodies for detection. Can also try interspersing with His with G/S residues. 50-100mM imidazole background binding works with this approach
A2: For mammalian and insect cells 8-10 His is the best tag length. Prepare membranes rather than directly solubilising cells. Reverse IMAC with tag cleavage is also useful.

Q: Could His tag bind SMA?
A1: SMI might be better for binding allowing more stringent washing.
A2: Charged residues among His tags could improve binding. Polar residue spacers can be good.

Q: Does anyone have experience of nanogold labelling via the His tag in protein-SMALPs? Doesn’t seem to be working, even if His tag purification worked.
A: Probably a problem with the His tag. Could His tag optimisation work?

Q: Can other affinity tags be used?
A: FLAG tags could bind after removal of free SMA from samples. Release from resin using anti-FLAG peptide is slower with protein-SMALPs than other systems.

Q: Is ion exchange possible?
A: In our experience, everything sticks to the columns so no separation is achieved! Maybe would work with big proteins in which the protein charge dominates rather than the SMA charge.

Q: What are the best methods for polymer and SMALP quantification? Including lipid-only SMALPs?
A1: Use the absorbance properties of the styrene to quantify polymer
A2: Mass photometry could be used.

Polymer development

Q: Can biotin be incorporated into polymers for binding studies/surface immobilisation/pulldowns from solution.
A: Biotin-tagged copolymers are in development.

Q: Are there other tags or polymers that would be useful?
A1: His tags on polymers
A2: Fluorophores
A3: Nanogold labels
A4: Isotopically labelled polymers: deuterated polymers for neutron scattering.
A5: Thiolated polymers (protocols exist for producing SMASH in the lab, see publications list).

Nanodisc size

Q: I wonder to use SMALP or DIBMA co-polymer to solubilize the protein complex which diameter is between 20~30nm. Which product is suitable for this case?
A1: DIBMA generally produce larger nanodiscs compared to SMA to my knowledge.
A2: I've had good success with DIBMA for larger complexes, approx 1:1 to the lipid content. But this was with purified lipid and structure.
A3: Substituted polymers with reduced charge, including those that are zwitterionic, will tend to produce larger discs presumably due to reduce repulsive forces.

Polymer concentration

Q: I would like to know the concentration of SMA in the solution?
A1: DIBMA generally produce larger nanodiscs compared to SMA to my knowledge.
A2: I've had good success with DIBMA for larger complexes, approx 1:1 to the lipid content. But this was with purified lipid and structure.
A3: We tend to start with 2.5% concentration and titrate down depending on the protein and downstream applications
A4: I just do a BCA assay to determine the concentration
A5: You can quantify it with a PDA detector

Polymer stability

Q: I’m curious about the light sensitivity of the powder. Has anyone has problems with stocks going bad?
A1: It is generally recommended to store activated (hydrolyzed) polymer in the dark and in the fridge. See also the Workman et al. 2022 paper.

Contact us to enquire about collaborative interactions or contract services, or share new protocols for generating improved co-polymer solutions and stable nanoparticles containing native membrane:protein complexes or "memteins" for structure function analysis.

Cryo-EM structure of AcrB trimer in a section of native bilayer as determined by Qiu et al and reviewed by Overduin et al.