TITLE: Purification of Lactococcus lactis OpuA and reconstitution in MSP nanodiscs
AUTHORS: Marco van den Noort, Panagiotis Drougkas, Cristina Paulino, Bert Poolman

[DESCRIPTION]
This is an optimized purification protocol which has been optimized in the context of:
Van Den Noort, M., Drougkas, P., Paulino, C., and Poolman, B. The substrate-binding domains of the osmoregulatory ABC importer OpuA physically interact. eLife 12:RP90996 https://doi.org/10.7554/eLife.90996.1
 
 
Lactococcus lactis OpuA is a type I ABC importer. It employs two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver it to the transmembrane domains for transport. OpuA detects osmotic stress through alterations in internal ionic strength and is further modulated by the second messenger cyclic-di-AMP. It exists as a tetrameric protein complex composed of two distinct subunits: two cytosolic OpuAA proteins featuring a nucleotide-binding domain and a cyclic-di-AMP-sensing CBS domain, and two OpuABC proteins comprising a transmembrane domain, a scaffold domain, and an extracellular single substrate-binding domain. The oligomeric assembly exhibits modest stability, with a notable portion of OpuA complex dissociating during purification. Consequently, modifications were made to the purification protocol across various parameters to minimize OpuA complex dissociation.

[BEFORE_START]
Note that the protocol starts with OpuA-overexpressing cells as described before (https://doi.org/10.7554/eLife.90996.1 ; https://doi.org/10.1126/sciadv.abd7697).
 
In order to obtain a homogenous nanodisc sample, which consists of a tetrameric OpuA complex in a lipid-filled nanodisc, the previous protocol in our lab was optimized on several aspects. Once OpuA is solubilized with 0.5% DDM (w/w) in the presence of 20% glycerol (v/v) it is necessary to prevent dissociation of the complex [1]. Therefore, both the vesicle solubilization step and the incubation of the sample with the Ni-Sepharose resin were shortened to 30 min. Based on the shape of the size-exclusion chromatography (SEC) profile this new procedure reduced loss of OpuAA subunits (Figure 1). Knowing that the OpuA complex is not very stable and that high pressure lysing of cells exerts stress on proteins as well, 20% glycerol (v/v) was added during the preparation of membrane vesicles. Again, on the basis of the SEC profile this markedly reduced the loss of OpuAA subunits (Figure 2). 
            Shaking or agitation imposes potentially harmful air-water interface forces on proteins. Therefore, agitation during membrane vesicle solubilization and Ni-Sepharose binding was prevented in the new method. Also, less imidazole was used for the elution of the OpuA complex. Furthermore, we removed the His-tag from the MSP proteins, utilizing the TEV-cleavage site that is located between MSP-protein and the His-tag. We removed empty nanodiscs by an extra IMAC step after overnight reconstitution.
            For the preparation of nanodiscs we switched from MSP1D1 to MSP1E3D1, because cryoEM structures indicate that there is limited space for lipids around OpuA in MSP1D1 nanodiscs [2]. Previously, the synthetic lipid mixture was solubilized prior to reconstitution by extrusion through a 400 nm filter and subsequent vortexing in the presence of 12 mM DDM. The lipids were diluted to the proper concentration in the reconstitution mixture, but the DDM concentration was kept at 12 mM. A high DDM concentration during reconstitution induce dissociation of OpuA complexes (Figure 3, 4). Therefore, in the new method no extra DDM was added when the lipids were diluted in the reconstitution mixture. Consequently, the DDM concentration in the reconstitution mixture was less than 8 mM.
            During previous reconstitutions, the molar ratio of OpuA:MSP:lipid was 1:20:1000. However, after extrusion through a 400 nm filter and vortexing of the synthetic lipids in the presence of 12 mM DDM, the solution still appeared turbid. This could be an indication for the presence of unsolubilized liposomes. Those lipids will not participate in the reconstitution process and can contribute to heterogeneity in the final nanodisc sample. In the current method, the lipids are sonicated in 50 mM KPi pH 7.0 at a concentration of 6.25 mg/mL. Afterwards, the sample is incubated for at least 2 hours with 1% DDM (w/w). The lipid sample becomes fully transparent before it is used for the nanodisc reconstitution of OpuA. 
            Because most, if not all, lipids are solubilized with this new method and another membrane scaffolding protein (MSP1E3D1) is used, the molar ratio of OpuA:MSP:lipid was re-optimized. Since a OpuA:MSP ratio of 1:20 created a lot of empty nanodiscs, the ratio was changed to 1:10. The OpuA:MSP:lipid ratio of 1:10:200 gave the best SEC profile in terms of monodispersity and width of the SEC profile (Figure 5). For a short overview of the optimization steps, see table below.
 
 
  
 
 
 
 
 
  
 
 
 
 
 
  
  
 
 
 
REFERENCES
[1]      E. Biemans-Oldehinkel, B. Poolman, On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA, EMBO J. 22 (2003) 5983–5993. https://doi.org/10.1093/emboj/cdg581.
[2]      H.R. Sikkema, M. van den Noort, J. Rheinberger, M. de Boer, S.T. Krepel, G.K. Schuurman-Wolters, C. Paulino, B. Poolman, Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA, Sci. Adv. 6 (2020) eabd7697. https://doi.org/10.1126/sciadv.abd7697.
[3]      A. Drozdetskiy, C. Cole, J. Procter, G.J. Barton, JPred4: a protein secondary structure prediction server, Nucleic Acids Res. 43 (2015) W389–W394. https://doi.org/10.1093/nar/gkv332.

[STEPS]
SECTION: Preparation of crude membrane vesicles
1. Use cells with an OD600 of 150-200, which are resuspended in 50 mM KPi pH 7.5 plus 20% (v/v) glycerol. Perform all steps of this procedure on ice or in a cooled environment (4 to 8 °C), unless otherwise indicated.
SECTION: Preparation of crude membrane vesicles
2. Add 100 μg/ml DNAse plus 2 mM MgSO4. Also slowly add 1 mM PMSF, dissolved in isopropanol, while stirring the cell suspension.
SECTION: Preparation of crude membrane vesicles
3. Rupture the cells by passing them through a cooled (4 °C) high-pressure lyser HPL6 (Maximator GmbH) at a pressure of 30 kPsi. Repeat this step a second time.
SECTION: Preparation of crude membrane vesicles
4. Add 5 mM of EDTA and centrifuge at 4 °C for 25 min, 15,000 xg in Beckman J-20XP, using the JA-25.50 rotor.
SECTION: Preparation of crude membrane vesicles
5. Transfer the supernatant to Type 45 Ti or Type 50.2 Ti Bottle Assembly tubes by pouring (NB: The pellet can easily come loose. When pellet material comes along during the transfer of the supernatant, repeat the previous centrifuge step).
SECTION: Preparation of crude membrane vesicles
6. Centrifuge at 4 °C for 135 min, 42,000 rpm in Beckman Optima XE-90, using the Type 45 Ti rotor (138,298 xg at rav); or at 4 °C for 75 min, 45,000 rpm in Beckman Optima XE-90, using the Type 50.2 Ti rotor (184,842 xg at rav).
SECTION: Preparation of crude membrane vesicles
7. Remove the supernatant by pouring and release the pellet from the tube by using a pestle from a potter tube and a few mL of 50 mM KPi pH 7.5 plus 20% glycerol (v/v).
SECTION: Preparation of crude membrane vesicles
8. Pour the pellet material in a potter tube and wash the centrifuge tube at least two times with mM KPi pH 7.5 plus 20% glycerol (v/v) to retrieve all the material.
SECTION: Preparation of crude membrane vesicles
9. Fill the potter tube with extra buffer to obtain a volume that is equal to approximately 50% of the lysate after disruption, and resuspend the membranes completely by using the pestle of the potter tube.
SECTION: Preparation of crude membrane vesicles
10. Repeat step 5-8, but now use only 6 mL of 50 mM KPi (pH 7.5) plus 20% (v/v) glycerol per 100 mL of cells with an OD600 of 150-200 (volume of the cells at the start of the procedure) to resuspend the membrane vesicles.
SECTION: Preparation of crude membrane vesicles
11. Determine the protein concentration by means of a Pierce BCA assay. The expected concentration is 8-16 mg/mL.
SECTION: Preparation of crude membrane vesicles
12. Divide the sample in aliquots of 18 mg membrane vesicles, flash freeze the aliquots in liquid nitrogen and store them at -80 °C.
SECTION: Solubilization of synthetic lipids
13. Thaw 250 μL of 25 mg/mL lipids in 50 mM KPi pH 7.0. Dilute the lipids, using the same buffer, to 25 mg/mL in a 15 mL Falcon tube at room temperature.
SECTION: Solubilization of synthetic lipids
14. Tip-sonicate the lipids in an ice-water bath for 8 cycles of 15 sec with a 45 sec interval at an amplitude of 77 µm. For this synthetic lipid mix, tip-sonication is necessary to create small unilamellar vesicles, because DDM is not able to fully solubilize multilamellar vesicles within a time period of less than 5 h.
SECTION: Solubilization of synthetic lipids
15. Transfer 900 μL of the sonicated lipids to a small glass vial, and incubate at room temperature with 100 μL of 10% DDM (w/w) until the lipids are needed for OpuA reconstitution in lipid nanodiscs.
SECTION: Solubilization of crude membrane vesicles
16. Thaw the crude membrane vesicles at room temperature but put them on ice as soon as they are thawed.
SECTION: Solubilization of crude membrane vesicles
17. Dilute the crude membrane vesicles on ice in MLA-80 centrifuge tubes to 3 mg/mL in 6 mL. The final mixture should contain 50 mM KPi pH 7.0-7.5, 20% (v/v) glycerol, 200 mM KCl plus 0.5% (w/w) DDM. As an example, 9 mg/mL membrane vesicles are diluted according to the pipetting scheme below:
 
 
 
SECTION: Solubilization of crude membrane vesicles
18. Incubate the mixture for 30 min on ice and mix once by pipetting after 15 min. The sample is not mixed by agitation, because this induces dissociation of OpuAA subunits from the full OpuA complex.
SECTION: Solubilization of crude membrane vesicles
19. Centrifuge at 4 °C for 20 min, 80,000 rpm in Beckman MAX-E, using the MLA-80 rotor (336,896 xg at rav).
SECTION: Purification (Ni2+-Sepharose affinity chromatography)
20. Mix the supernatant in a 10 mL disposable chromatography column with 10 mM imidazole pH 7.5, and 0.5 mL of Ni2+-Sepharose (bed volume) that was washed with 12 column volumes of MQ and equilibrated with 4 column volumes of 50 mM KPi pH 7.0, 200 mM KCl, 20% (v/v) glycerol, 0.04% (w/w) DDM (Buffer A) plus 10 mM imidazole. Mix by gentle pipetting with a 1 mL pipette and make sure that the outlet of the column is closed.
SECTION: Purification (Ni2+-Sepharose affinity chromatography)
23. Elute OpuA by adding consecutively one time 0.6 and four times 0.4 column volumes of buffer A plus 200 mM imidazole pH 7.5. Collect the five fractions separately. Most protein is expected in the third and fourth fraction.
SECTION: Purification (Ni2+-Sepharose affinity chromatography)
22. Drain the column once the Ni2+-Sepharose is sedimented again and wash twice with 10 column volumes of buffer A plus 50 mM imidazole pH 7.5.
SECTION: Purification (Ni2+-Sepharose affinity chromatography)
21. Allow the Ni2+-Sepharose to sediment in 10-20 min, before mixing the sample a second time. The sample is not mixed by agitation, because this induces dissociation of OpuAA subunits from the full OpuA complex.
SECTION: Nanodisc reconstitution and second purification
24. Mix the following components in a 1.5 mL Eppendorf tube to an end volume of 700-900 μL and agitate for 1 hour at 4 °C: 
 
Note that this agitation step is necessary for nanodisc reconstitution but not ideal for OpuA. Therefore, try to minimize the empty volume in the tube.
SECTION: Nanodisc reconstitution and second purification
25. Add 500 mg of semi-dry activated SM-2 Bio-Beads to the sample and incubate overnight under gentle agitation at 4 °C.
SECTION: Nanodisc reconstitution and second purification
26. Separate the nanodiscs from the beads by using a syringe with a needle (⌀ 0.2 mm). To prevent transfer of beads that are stuck inside the needle, remove the needle before transferring the sample from the syringe in a new 1.5 mL Eppendorf tube.
SECTION: Nanodisc reconstitution and second purification
27. Spin down large particles at 20,817 xg for 15 min at 4°C. It is possible to directly proceed to size-exclusion chromatography if the resolution of the column is good enough to separate empty nanodiscs from the nanodiscs that contain the protein of interest. In this study, point 28-31 are mainly used for maleimide based cysteine labeling. 
SECTION: Nanodisc reconstitution and second purification
28. Pipette the supernatant gently to a 2 mL disposable chromatography column, which contains 0.2-0.3 mL of Ni2+-Sepharose (bed volume) that was washed with 10 column volumes of MQ and equilibrated with 5 column volumes of 50 mM KPi pH 7.0.
SECTION: Nanodisc reconstitution and second purification
29. Let the sample flow through, collect the flow-through as a single fraction and reapply it twice back to the Ni2+-Sepharose.
SECTION: Nanodisc reconstitution and second purification
30. Wash two times with 5 column volumes of 20 mM HEPES-K pH 7.0, 300 mM KCl plus 25 mM imidazole pH 7.5.
SECTION: Nanodisc reconstitution and second purification
31. Elute OpuA by adding consecutively 0.8 and 2.5 column volumes of 20 mM HEPES-K pH 7.0, 300 mM KCl plus 200 mM imidazole pH. Collect the two fractions separately.
SECTION: Size-exclusion chromatography and storage
32. Load the second elution fraction on a SuperdexTM 200, 10/300GL Increase column, preequilibrated with 20 mM HEPES-K pH 7.0 plus 300 mM KCl, and collect the elution in 0.5 mL fractions. 
SECTION: Size-exclusion chromatography and storage
33. Take the two best fractions containing OpuA nanodiscs, flash freeze them in liquid nitrogen in 50-100 μL aliquots and store them at -80 °C.