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Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies
生成、纯化和结晶原核生物中SLC26同源蛋白用于结构研究   

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Abstract

The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.

Keywords: Membrane transport protein(膜转运蛋白), Nanobody(纳米抗体), Crystallization chaperone(结晶伴侣), Solute carrier(溶质载体), SLC26(SLC26)

Background

With few exceptions, structural characterization of membrane proteins involves challenges at the level of protein production, stabilization in the detergent-solubilized state, and crystallization. The strategy we have followed to overcome these hurdles relied on the efficient selection of SLC26 homologs with superior biochemical properties and the use of antibodies as crystallization chaperones (Geertsma et al., 2015). The procedures described here do not greatly deviate from those of colleagues, but on a few points we do follow alternative approaches. For example, for protein production we make use of the araBAD promoter (Guzman et al., 1995) and not the popular T7 promoter (Studier et al., 1990). In contrast to the T7 promoter, the PBAD promoter allows direct tuning of the protein production levels and its adjustment to the capacity of the downstream folding machinery, thereby reducing the formation of inclusion bodies (Geertsma et al., 2008). Furthermore, we prefer nanobodies, the variable domain of camelid heavy chain only antibodies (Pardon et al., 2014), as crystallization chaperones over the more commonly used Fabs. In our hands, the generation, selection, and production of nanobodies is far more robust and straightforward. Though we are aware that alternative protein production strategies (Henderson et al., 2000; Kunji et al., 2003; Miroux and Walker, 1996; Studier, 2005; Wagner et al., 2008) and crystallization chaperones (Koide, 2009; Seeger et al., 2013) exist, we did not explore these as the presented procedures proved very robust and successful.

Materials and Reagents

  1. Dialysis tube
    MWCO 8 kDa (Carl Roth, catalog number: 1924.1 )
    MWCO 3.5 kDa (Carl Roth, catalog number: E860.1 )
  2. Concentrators
    MWCO 50 kDa (EMD Millipore, catalog number: UFC905024 )
    MWCO 3 kDa (EMD Millipore, catalog number: UFC800324 )
  3. Crystallization plates (HAMPTON RESEARCH, catalog number: HR3-158 )
  4. Potter tube and piston (VWR, catalog numbers: 432-0205 and 432-0211 )
  5. 50 ml tube
  6. E. coli MC1061 (Coli Genetic Stock Center, catalog number: 6649 )
  7. Plasmid pBXNPHM3-Nb5776 (Figure 1A)
    Note: This plasmid holds the gene coding for the nanobody under control of the araBAD promoter and results in the production of the nanobody fused to an N-terminal pelB leader sequence followed by decaHis-tag, MBP, and a HRV 3C protease site. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.
  8. Plasmid pBXC3GH-SLC26Dg (Figure 1B)
    Note: This plasmid holds the gene coding for SLC26Dg under control of the araBAD promoter and results in the production of SLC26Dg fused to a C-terminal HRV 3C protease site, GFP, and a decaHis-tag. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.



    Figure 1. Plasmid maps. A. Plasmid map of pBXNPHM3-Nb5776; B. Plasmid map of pBXC3GH-SLC26Dg. Unique restriction sites and important features in the plasmids are indicated.

  9. Ampicillin sodium salt (Carl Roth, catalog number: K029.2 )
  10. L-arabinose (20% w/v in ddH2O) (Carl Roth, catalog number: 5118.2 )
  11. 1 M KPi, pH 7.5 (see Recipes)
    K2HPO4(AppliChem, catalog number: 121512 )
    KH2PO4(AppliChem, catalog number: 131509 )
  12. Sodium chloride (NaCl, 2.5 M, ddH2O) (Carl Roth, catalog number: 3957.1 )
  13. Lysozyme (100 mg/ml in ddH2O) (AppliChem, catalog number: A3711 )
  14. DNAse I (2 mg/ml in ddH2O) (AppliChem, catalog number: A3778 )
  15. Magnesium sulphate heptahydrate (MgSO4, 1 M, ddH2O) (Carl Roth, catalog number: P027.2 )
  16. Phenylmethyl sulphonyl fluoride (PMSF, 200 mM in ethanol) (Carl Roth, catalog number: 6367.1 )
  17. NiNTA (50% w/v slurry in 20% EtOH) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88223 )
  18. ddH2O
  19. Imidazole (2 M, pH 7.5, HCl, ddH2O) (Carl Roth, catalog number: X998.4 )
  20. HEPES (1 M, pH 7.5, NaOH, ddH2O) (Carl Roth, catalog number: HN78.2 )
  21. HRV 3C protease (homemade)
  22. Polypropylene glycol 2000 (10% v/v in ddH2O) (Sigma-Aldrich, catalog number: 81380-1L )
  23. EDTA, disodium salt dihydrate (Carl Roth, catalog number: X986.1 )
  24. Glycerol, 86% (w/v) (Carl Roth, catalog number: 4043.3 )
  25. Liquid nitrogen
  26. Decylmaltoside (10% w/v in ddH2O) (Anatrace, catalog numbers: D322S and D322LA )
  27. Ammonium formate (Carl Roth, catalog number: 5093.1 )
  28. Sodium acetate (Na-acetate) (Carl Roth, catalog number: 6773.2 )
  29. PEG400 (Sigma-Aldrich, catalog number: 91893 )
  30. Tryptone (AppliChem, catalog number: A1553 )
  31. Yeast extract (AppliChem, catalog number: A1552 )
  32. Agar (Applichem, catalog number: A0917 )
  33. LB agar (see Recipes)
  34. TB medium (see Recipes)
  35. Reservoir solution (see Recipes)

Equipment

  1. 5 L baffled flask
  2. Incubator at 37 °C
  3. Shaker with adjustable temperature (Infors, model: HT Multitron )
  4. Centrifuge for pelleting cultures (Thermo Fisher Scientific, Thermo Scientific, model: Thermo Sorvall Evolution RC )
  5. Homogenizer (IKA, model: ULTRA-TURRAX® T25 )
  6. High pressure cell disrupter (Avestin, model: Emulsiflex C3 )
  7. Nanodrop (Thermo Fisher Scientific, NanodropTM, model: 1000 , discontinued)
  8. Magnet stirrer (Heidolph Instruments, model: Hei-Mix S )
  9. SEC column Superdex 75 10/300 GL (GE Healthcare, catalog number: 17-5174-01 )
  10. SEC column Superdex 200 10/300 GL (GE Healthcare, catalog number: 28-9909-44 )
  11. Table-centrifuge (VWR, model: Micro Star 17 )
  12. HPLC (GE Healthcare, model: ÄKTAprime plus )
  13. Fermenter (BIOENGiNEERiNG, model: NLF 22, 30 L)
  14. Spectrophotometer (Amersham Biosciences, model: Ultrospec 10 )
    Note: This product has been discontinued.
  15. Ultracentrifuge (Beckman Coulter, model: Optima XPN-100K Ultracentrifuge )

Procedure

  1. Production and purification of Nb5776
    1. Transform E. coli MC1061 with pBXNPHM3-Nb5776. Plate on LB-agar plates containing 100 μg/ml ampicillin. Grow overnight at 37 °C.
    2. Pick a single colony to inoculate a preculture of 20 ml TB medium supplemented with 100 μg/ml ampicillin (TB/Amp). Grow overnight at 37 °C.
    3. Inoculate 1.5 L TB/Amp with 15 ml preculture in a 5 L baffled flask and cultivate at 37 °C. Adjust shaking speed to guarantee sufficient aeration while avoiding foam formation (~100 rpm).
    4. After 1.5 h set the temperature of the incubator to 25 °C and allow the culture to cool over the course of 1 h. Induce with a final concentration of 0.01% L-arabinose once the OD600 is between 0.5-1.0. Continue incubation overnight (~16 h).
    5. Harvest the cells by centrifugation at 5,500 x g for 15 min at 4 °C. From here on keep the sample cool (0-4 °C) and precool solutions and equipment beforehand.
    6. Resuspend the cells in ice-cold 50 mM KPi, pH 7.5, 150 mM NaCl to a final OD600 = 150-200.
    7. Add lysozyme (1 mg/ml final), DNAse I (20 μg/ml final concentration), and MgSO4 (1 mM final concentration).
    8. Homogenize the cells, e.g., using an Ultra-Turrax T25 homogenizer.
    9. Incubate for ~60 min at 4 °C while stirring.
    10. Disrupt cells by three passes at 10 kPsi using a high pressure cell disrupter (Emulsiflex, precooled).
    11. Add PMSF to a final concentration of 1 mM.
    12. Centrifuge the lysate for 30 min at 140,000 x g at 4 °C.
    13. Prepare the NiNTA column. Use 3 ml solid NiNTA. Wash the column with ~10 column volumes (CV; 30 ml) ddH2O and with ~10 CV (30 ml) 50 mM KPi, pH 7.5, 150 mM NaCl.
    14. Transfer the supernatant from step A12 to a new container and supplement with imidazole to a final concentration of 15 mM.
    15. Incubate the supernatant with NiNTA for 1 h at 4 °C, rotating.
    16. Drain column.
    17. Wash column with 20 CV (60 ml) 20 mM HEPES, pH 7.5, 500 mM NaCl, 50 mM imidazole.
    18. Elute column with 5x 0.5 CV (1.5 ml) 20 mM HEPES, pH 7.5, 150 mM NaCl, 300 mM imidazole.
    19. Determine protein concentration using a Nanodrop.
    20. Pool peak fractions and add 3C protease to a final protein:HRV 3C protease molar ratio of 5:1.
    21. Digest overnight at 4 °C while dialyzing against 20 mM HEPES, pH 7.5, 150 mM NaCl. Use a stirrer bar in the dialysis buffer. The MWCO of the dialysis tube should be 3.5 kDa. The dialysis volume should be sufficient to obtain a final imidazole concentration of 15 mM upon complete equilibration.
    22. Equilibrate the size-exclusion chromatography column (Superdex 75 10/300 GL) with 2.5 CV 10 mM HEPES, pH 7.5, 150 mM NaCl.
    23. Incubate the dialyzed material with 1 ml solid NiNTA (prewashed with ddH2O and dialysis buffer as in step A13) for 10 min at 4 °C, rotating.
    24. Collect the flow-through in a concentrator (3 kDa MWCO). The concentrator should be pre-run with ddH2O and flushed with dialysis buffer. Wash the NiNTA column with 3 CV dialysis buffer and collect this material in the concentrator as well.
    25. Centrifuge the concentrator for < 10 min at ~3,000 x g, 4 °C. Take it out and invert a few time to homogenize the solution (alternatively: pipet it up and down). Repeat until desired volume is reached (< 0.5 ml).
    26. Transfer sample to a precooled 1.5 ml cup. Centrifuge for 10 min at 16,000 x g in a table centrifuge at 4 °C.
    27. Transfer supernatant to a new precooled cup.
    28. Prepare the HPLC. Place new tubes in the fraction collector. Take the injection syringe apart and wash it with dialysis buffer. Remove bubbles from the syringe by rapidly pushing out the liquid. Pre-flush the needle and loop with dialysis buffer.
    29. Inject the sample into the loop. Start the run. Once the peak is approaching, change the fractionation volume to 0.3 ml. After the peak change to 1.4 ml fractionation.
    30. Determine which fractions contain the peak based on the chromatogram. Determine the protein concentration in these fractions using the Nanodrop.
    31. Pool peak fractions and concentrate (3 kDa MWCO) to a final concentration over 10 mg/ml.
    32. Store the sample at 4 °C until use. Nb5776 remains stable for at least 3 months.

  2. Production and purification of SLC26Dg
    1. Transform E. coli MC1061 with pBXC3GH_SLC26Dg. Plate on LB-agar plates containing 100 μg/ml ampicillin. Grow overnight at 37 °C.
    2. Pick a single colony to inoculate a preculture of 100 ml TB/Amp. Grow overnight at 37 °C.
    3. Inoculate 9 L TB/Amp with 90 ml preculture in a fermenter and cultivate at 37 °C. Adjust stirring and aeration to guarantee rapid growth. Adjust vessel pressure to approximately 1 bar overpressure to reduce foaming and supplement culture with small aliquots of a 10% (v/v) solution of polypropylene glycol 2000 if foam is formed. Continue cultivation until an OD600 of approximately 2 is reached.
    4. Gradually reduce the temperature to 25 °C over the course of 1 h. After an additional 20 min, induce the culture with 2.5 ml 20% (w/v) L-arabinose. Continue incubation overnight.
    5. Harvest the cells by centrifugation at 5,500 x g for 15 min at 4 °C. From here on keep the sample cool (0-4 °C) and precool solutions and equipment beforehand.
    6. Resuspend the cells in 50 mM KPi, pH 7.5, 150 mM NaCl to an OD600 = 150-200.
    7. Add lysozyme (1 mg/ml final), DNAse I (20 μg/ml final concentration), and MgSO4 (1 mM final concentration).
    8. Homogenize the cells, e.g., using an Ultra-Turrax T25 homogenizer.
    9. Incubate for ~60 min at 4 °C while stirring.
    10. Disrupt cells by three passes at 10 kPsi using a high pressure cell disrupter (Emulsiflex, precooled).
    11. Add PMSF (1 mM final concentration) and EDTA (5 mM final concentration).
    12. Perform a low spin centrifugation to remove unbroken cells, 15 min at 10,000 x g at 4 °C.
    13. Transfer supernatant to tubes suitable for ultracentrifugation.
    14. Perform a high spin centrifugation to pellet the membranes, 1 h at 160,000 x g at 4 °C.
    15. Determine the net-weight of the vesicle pellets.
    16. Resuspend the vesicles to ~1 g/2 ml in 50 mM KPi, pH 7.5, 150 mM NaCl, 10% glycerol using a Potter tube.
    17. Freeze the vesicles in liquid nitrogen in aliquots of 1/5/10 g.
    18. Store the aliquots at -80 °C until use. Vesicles can be stored under this condition over 1 year.
    19. Thaw 5 g membrane vesicles in a beaker with room temperature water and a stirrer bar. Keep the sample on ice once it is thawed.
    20. Equilibrate the decylmaltoside (sol-grade) powder to room temperature.
    21. Transfer the viscous vesicle suspension to a 50 ml tube. Wash the tube with ice-cold 50 mM KPi, pH 7.5, 150 mM NaCl, 10% glycerol and adjust the volume to 40 ml.
    22. Add imidazole to a final concentration of 15 mM.
    23. Add 0.625 g decylmaltoside to give a final concentration of ~1.6%. It is important to maintain a vesicles:detergent ratio of approximately 5 g:0.5-0.6 g in order to get good solubilization.
    24. Incubate 1 h at 4 °C, rotating.
    25. Centrifuge the material for 30 min at 160,000 x g at 4 °C.
    26. Prepare the NiNTA column. Use 3 ml solid NiNTA. Wash the column with ~10 CV (30 ml) ddH2O and with ~10 CV (30 ml) 50 mM KPi, pH 7.5, 150 mM NaCl, 10% glycerol.
    27. Incubate the supernatant with NiNTA for 1 h at 4 °C, rotating.
    28. Drain column.
    29. Wash column with 20 CV (60 ml) 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 50 mM imidazole, 0.2% decylmaltoside.
    30. Elute column with five aliquots of 0.5 CV (1.5 ml) 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 300 mM imidazole, 0.2% decylmaltoside.
    31. Determine protein concentration using the Nanodrop.
    32. Pool peak fractions and add 3C protease to a final protein:3C protease molar ratio of 5:1.
    33. Digest for 2 h at 4 °C while dialyzing against 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.13% decylmaltoside. Use a stirrer bar in the dialysis buffer. The MWCO of the dialysis tube should be 8 kDa. The dialysis volume should be sufficient to obtain a final imidazole concentration of 15 mM upon complete equilibration.
    34. Equilibrate the size-exclusion chromatography column (Superdex 200 10/300 GL) with 2.5 CV 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.2% decylmaltoside (highest purity).
    35. Incubate the dialyzed material with 0.6 ml solid NiNTA (prewashed with ddH2O and dialysis buffer as in step B26) for 10 min at 4 °C, rotating.
    36. Collect the flow-through in a concentrator (50 kDa MWCO). The concentrator should be pre-run with ddH2O and flushed with dialysis buffer. Wash the NiNTA column with 3 CV dialysis buffer and collect this flow-through in the concentrator as well.
    37. Centrifuge the concentrator for < 10 min at 3,000 x g, 4 °C. Take it out and invert a few time to homogenize the solution (alternatively: pipet it up and down). Repeat until desired volume is reached (< 0.5 ml).
    38. Transfer sample to a precooled 1.5 ml cup. Centrifuge for 10 min at > 16,000 x g in a table centrifuge at 4 °C.
    39. Transfer supernatant to a new precooled cup.
    40. Prepare the HPLC. Place new tubes in the fraction collector. Take the injection syringe apart and wash it with dialysis buffer. Remove bubbles from the syringe by rapidly pushing out the liquid. Pre-flush the needle and loop with dialysis buffer.
    41. Inject the sample into the loop. Start the run. Once the peak is approaching, change the fractionation volume to 0.3 ml. After the peak change to 1.4 ml fractionation.
    42. Determine which fractions contain the peak based on the chromatogram. Determine the protein concentration in these fractions using the Nanodrop.
    43. Pool peak fractions and concentrate (50 kDa MWCO) to a final concentration over 10 mg/ml.
    44. Do not store the sample but immediately proceed with complex formation and crystallization.

  3. Complex formation and crystallization of SLC26Dg-Nb5776
    1. Supplement purified and concentrated Nb5776 with decylmaltoside (10% w/v stock) to a final concentration of 0.2% (w/v).
    2. Mix purified SLC26Dg and Nb5776 at a molar ratio of 1:2. Incubate for 10 min at 4 °C.
    3. Submit the sample to SEC using a Superdex 200 10/300 GL column equilibrated with 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.2% decylmaltoside.
    4. Determine which fractions contain the peak based on the chromatogram. Determine the protein concentration in these fractions using the Nanodrop. Verify the presence of both proteins in the fractions by SDS-PAGE.
    5. Pool peak fractions and concentrate (50 kDa MWCO) to a final concentration of approximately 9-12 mg/ml.
    6. Grow SLC26Dg-Nb5776 crystals in sitting drops (1 μl protein + 1 μl reservoir solution [see Recipes]) by vapor diffusion at 4 °C. Mix protein and reservoir solutions in a 1:1 ratio. Crystals appear after approximately 1 week.

Data analysis

Representative data is depicted in Figures 2 and 3.


Figure 2. Purification of SLC26Dg. SDS-PAGE analysis of samples obtained at different steps during the purification of SLC26Dg according to the protocol detailed here. ‘Elution’, ‘digested’, and ‘SEC’, refer to samples obtained after elution from the NiNTA column, after digestion with HRV 3C protease, and after elution from the SEC column, respectively. Black and white arrows indicate bands containing GFP (fused to SLC26Dg or alone) and SLC26Dg, respectively. The right panel represents an image of the SDS-PAGE gel obtained by measuring the in gel GFP fluorescence. The left panel represents a coomassie-stained image of the same gel. Molecular weight markers (in kDa) are indicated on the left.



Figure 3. Complex formation of SLC26Dg and Nb5776. A. Size-exclusion chromatogram of SLC26Dg pre-incubated with an excess of Nb5776 as indicated in the protocol. B. SDS-PAGE analysis of the relevant peak fractions from the SEC column. C. Protein crystal of the SLC26Dg-Nb5776 complex.

Recipes

  1. LB agar (1 L)
    10 g tryptone
    5 g yeast extract
    5 g NaCl
    15 g agar
    Add demineralized H2O to 1 L and autoclave at 121 °C for 20 min
  2. TB medium (1 L)
    1. 12 g tryptone
      24 g yeast extract
      8.6 ml 86% (w/v) glycerol
      Add demineralized H2O up to 950 ml
    2. 50 ml 1.44 M K2HPO4, 0.34 M KH2PO4 in ddH2O
      Autoclave both solutions separately (at 121 °C for 20 min) and mix before use
  3. 1 M KPi, pH 7.5 (200 ml)
    Prepare a 200 ml solution of 1 M K2HPO4 and a 100 ml solution of 1 M KH2PO4 using ddH2O
    Mix both solutions in an approximate ratio of 4:1 (K2HPO4:KH2PO4) under constant stirring while measuring the pH
    Set the pH by adjusting with the basic (K2HPO4) or acidic (KH2PO4) component
  4. Reservoir solution
    1 M ammonium formate, 50 mM Na-acetate, pH 4.5
    45-50% PEG400 (w/v)

Acknowledgments

The protocols detailed here were developed in the laboratory of Prof. Raimund Dutzler at the University of Zurich and benefitted from helpful suggestions from Drs. Sandra Markovic, Ricarda J.C. Hilf, Ines Ehrnstorfer, Stefan Warmuth, Iwan Zimmermann, and Prof. Markus Seeger. Prof. Jan Steyaert and Dr. Els Pardon from the Vrije Universiteit Brussel are acknowledged for the generation and selection of Nb5776. Beat Blattman and Céline Stutz-Ducommun from the Protein Crystallization Center of the University of Zürich are acknowledged for their support in establishing crystallization conditions for SLC26Dg/Nb5776. E.R.G. acknowledges support by a long-term fellowship from the Human Frontier Science Program (LT-00899/2008) and the German Research Foundation through the Cluster of Excellence Frankfurt ‘Macromolecular Complexes’.

References

  1. Geertsma, E. R., Chang, Y. N., Shaik, F. R., Neldner, Y., Pardon, E., Steyaert, J. and Dutzler, R. (2015). Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nat Struct Mol Biol 22(10): 803-808.
  2. Geertsma, E. R., Groeneveld, M., Slotboom, D. J. and Poolman, B. (2008). Quality control of overexpressed membrane proteins. Proc Natl Acad Sci U S A 105(15): 5722-5727.
  3. Guzman, L. M., Belin, D., Carson, M. J. and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177(14): 4121-4130.
  4. Henderson, P. J., Hoyle, C. K. and Ward, A. (2000). Expression, purification and properties of multidrug efflux proteins. Biochem Soc Trans 28(4): 513-517.
  5. Koide, S. (2009). Engineering of recombinant crystallization chaperones. Curr Opin Struct Biol 19(4): 449-457.
  6. Kunji, E. R., Slotboom, D. J. and Poolman, B. (2003). Lactococcus lactis as host for overproduction of functional membrane proteins. Biochim Biophys Acta 1610: 97-108.
  7. Miroux, B. and Walker, J. E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260(3): 289-298.
  8. Pardon, E., Laeremans, T., Triest, S., Rasmussen, S. G., Wohlkonig, A., Ruf, A., Muyldermans, S., Hol, W. G., Kobilka, B. K. and Steyaert, J. (2014). A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9(3): 674-693.
  9. Seeger, M. A., Zbinden, R., Flutsch, A., Gutte, P. G., Engeler, S., Roschitzki-Voser, H. and Grutter, M. G. (2013). Design, construction, and characterization of a second-generation DARPin library with reduced hydrophobicity. Protein Sci 22(9): 1239-1257.
  10. Studier, F. W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1): 207-234.
  11. Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60-89.
  12. Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Hogbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O. and de Gier, J. W. (2008). Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105(38): 14371-14376.

简介

SLC26或SulP蛋白构成在亲和真核生物中普遍表达的大量阴离子转运蛋白。在人类中,SLC26蛋白在离子稳态中起重要作用,选择成员的功能障碍与疾病有关。该方案详细描述了来自地热异常球菌的原核SLC26同源物(称为SLC26Dg)的产生和结晶。按照这些说明,我们获得了膜蛋白SLC26Dg和纳米体Nb5776的良好折叠和均匀的材料,使我们能够使复合物结晶并确定其结构(Geertsma等人,2015)。该方法可以适于纯化和结晶其它膜蛋白复合物。

背景 除了少数例外,膜蛋白的结构表征涉及蛋白质生产水平,洗涤剂溶解状态下的稳定化和结晶的挑战。克服这些障碍所采取的策略取决于有效选择具有优异生物化学性质的SLC26同系物和使用抗体作为结晶伴侣(Geertsma等人,2015)。这里描述的程序并没有大大偏离同事的程序,但在几点上,我们采用其他方法。例如,对于蛋白质生产,我们利用araBAD启动子(Guzman等人,1995),而不是流行的T7启动子(Studier等人,1990)。与T7启动子相反,PAD启动子允许直接调节蛋白质生产水平及其对下游折叠机械的能力的调节,从而减少包涵体的形成(Geertsma, et al。,2008)。此外,我们更喜欢纳米体系,即骆驼重链仅抗体的可变结构域(Pardon等,2014),作为比较常用的Fabs的结晶分子伴侣。在我们手中,纳米碳体的生成,选择和生产更加强大和直接。虽然我们知道替代的蛋白质生产策略(Henderson等人,2000; Kunji等人,2003; Miroux和Walker,1996; Studier,2005; Wagner <存在,结晶分子伴侣(Koide,2009; Seeger等人,2013)存在,我们没有探索这些,因为呈现的程序证明非常强大和成功。

关键字:膜转运蛋白, 纳米抗体, 结晶伴侣, 溶质载体, SLC26

材料和试剂

  1. 透析管
    MWCO 8 kDa(Carl Roth,目录号:1924.1)
    MWCO 3.5kDa(Carl Roth,目录号:E860.1)
  2. 集中者
    MWCO 50 kDa(EMD Millipore,目录号:UFC905024)
    MWCO 3 kDa(EMD Millipore,目录号:UFC800324)
  3. 结晶板(HAMPTON RESEARCH,目录号:HR3-158)
  4. 波特管和活塞(VWR,目录号:432-0205和432-0211)
  5. 50ml管
  6. E。大肠杆菌MC1061(Coli Genetic Stock Center,目录号:6649)
  7. 质粒pBXNPHM3-Nb5776(图1A)
    注意:该质粒含有在araBAD启动子控制下编码纳米体的基因,并导致与N末端pelB前导序列融合的纳米体的产生,随后是decaHis标签,MBP和HRV 3C蛋白酶现场。它含有β-内酰胺抗生素标记。质粒可根据要求提供非商业用途。
  8. 质粒pBXC3GH-SLC26Dg(图1B)
    注意:该质粒将编码SLC26Dg的基因保持在araBAD启动子的控制下,并导致与C端HRV 3C蛋白酶位点,GFP和decaHis标签融合的SLC26Dg的产生。它含有β-内酰胺抗生素标记。质粒可根据要求提供非商业用途。



    图1.质粒图。 A。 pBXNPHM3-Nb5776质粒图; B. pBXC3GH-SLC26Dg的质粒图。指出了质粒的独特的限制性位点和重要特征
  9. 氨苄青霉素钠盐(Carl Roth,目录号:K029.2)
  10. L-阿拉伯糖(ddH 2 O中为20%w/v)(Carl Roth,目录号:5118.2)
  11. 1 M KPi,pH 7.5(见配方)
    K 2(HPO 4)(AppliChem,目录号:121512)
    KH 2 PO <4>(AppliChem,目录号:131509)
  12. 氯化钠(NaCl,2.5M,ddH 2 O)(Carl Roth,目录号:3957.1)
  13. 溶菌酶(ddH 2 O中为100mg/ml)(AppliChem,目录号:A3711)
  14. DNAse I(ddH 2 O中为2mg/ml)(AppliChem,目录号:A3778)
  15. 硫酸镁七水合物(MgSO 4,1M,ddH 2 O)(Carl Roth,目录号:P027.2)
  16. 苯基甲基磺酰氟(PMSF,乙醇中200mM)(Carl Roth,目录号:6367.1)
  17. NiNTA(50%w/v浆液在20%EtOH中)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88223)
  18. ddH 2 O
  19. 咪唑(2M,pH7.5,HCl,ddH2O)(Carl Roth,目录号:X998.4)
  20. HEPES(1M,pH7.5,NaOH,ddH 2 O)(Carl Roth,目录号:HN78.2)
  21. HRV 3C蛋白酶(自制)
  22. 聚丙二醇2000(ddH 2 O中为10%v/v)(Sigma-Aldrich,目录号:81380-1L)
  23. EDTA,二钠盐水合物(Carl Roth,目录号:X986.1)
  24. 甘油,86%(w/v)(Carl Roth,目录号:4043.3)
  25. 液氮
  26. 癸基麦芽糖苷(ddH 2 O中为10%w/v)(Anatrace,目录号:D322S和D322LA)
  27. 甲酸铵(Carl Roth,目录号:5093.1)
  28. 醋酸钠(醋酸钠)(Carl Roth,目录号:6773.2)
  29. PEG400(Sigma-Aldrich,目录号:91893)
  30. Trypton(AppliChem,目录号:A1553)
  31. 酵母提取物(AppliChem,目录号:A1552)
  32. 琼脂(应用,目录号:A0917)
  33. LB琼脂(参见食谱)
  34. TB培养基(参见食谱)
  35. 水库解决方案(见配方)

设备

  1. 5 L挡板烧瓶
  2. 孵化器在37°C
  3. 可调温度的振动筛(Infors,型号:HT Multitron)
  4. 离心机用于造粒培养(Thermo Fisher科学,Thermo科学,型号:Thermo Sorvall Evolution RC)
  5. 均质器(IKA,型号:ULTRA-TURRAX T25)
  6. 高压细胞破坏器(Avestin,型号:Emulsiflex C3)
  7. Nanodrop(Thermo Fisher Scientific,Nanodrop TM,型号:1000,停产)
  8. 磁力搅拌器(Heidolph Instruments,型号:Hei-Mix S)
  9. SEC栏Superdex 75 10/300 GL(GE Healthcare,目录号:17-5174-01)
  10. SEC列Superdex 200 10/300 GL(GE Healthcare,目录号:28-9909-44)
  11. 台式离心机(VWR,型号:Micro Star 17)
  12. HPLC(GE Healthcare,型号:ÄKTAprimeplus)
  13. 发酵罐(BIOENGiNEERiNG,型号:NLF22,30L)
  14. 分光光度计(Amersham Biosciences,型号:Ultrospec 10)
    注意:本产品已停产。
  15. 超速离心机(Beckman Coulter,型号:Optima XPN-100K Ultracentrifuge)

程序

  1. 生产和净化Nb5776
    1. 变换E。大肠杆菌MC1061与pBXNPHM3-Nb5776。在含有100μg/ml氨苄青霉素的LB-琼脂平板上培养。在37°C过夜生长。
    2. 挑取单个菌落以接种补充有100μg/ml氨苄青霉素(TB/Amp)的20ml TB培养基的预培养物。在37°C过夜生长。
    3. 在5L挡板烧瓶中接种1.5L TB/Amp,15ml预培养物,并在37℃下培养。调节振动速度以确保足够的通气,同时避免泡沫形成(〜100 rpm)。
    4. 1.5小时后,将培养箱的温度设定在25℃,使培养物在1小时内冷却。诱导0.01%L-阿拉伯糖的终浓度,一旦OD 600在0.5-1.0之间。继续孵育过夜(〜16 h)。
    5. 通过在4℃下以5,500×g离心15分钟收获细胞。从这里保持样品冷却(0-4°C)和预冷解决方案和设备预先。
    6. 将细胞重悬于冰冷的50mM KPi,pH7.5,150mM NaCl中至最终OD 600 = 150-200。
    7. 加入溶菌酶(最终1mg/ml),DNAse I(20μg/ml终浓度)和MgSO 4(最终浓度为1mM)。
    8. 使用Ultra-Turrax T25匀浆器使细胞均质化,例如。
    9. 在搅拌下,在4℃下孵育约60分钟。
    10. 使用高压细胞破坏器(Emulsiflex,预冷)以10 kPsi通过三次通过破坏细胞。
    11. 加入PMSF至最终浓度为1 mM。
    12. 在4℃以140,000×g离心裂解物30分钟。
    13. 准备NiNTA柱。使用3毫升固体NiNTA。用〜10柱体积(CV; 30ml)ddH 2 O和约10℃(30ml)的50mM KPi,pH7.5,150mM NaCl洗涤柱。
    14. 将上清液从步骤A12转移到新容器中,并用咪唑补充至终浓度为15mM
    15. 在4℃下,用NiNTA孵育上清1小时,旋转
    16. 排水柱。
    17. 用20 CV(60ml)20mM HEPES,pH7.5,500mM NaCl,50mM咪唑洗涤柱
    18. 具有5×0.5CV(1.5ml)20mM HEPES,pH7.5,150mM NaCl,300mM咪唑的洗脱柱。
    19. 使用Nanodrop确定蛋白质浓度
    20. 池峰分数并加入3C蛋白酶至最终蛋白质:HRV 3C蛋白酶摩尔比为5:1。
    21. 在4℃下消化过夜,同时对20mM HEPES,pH 7.5,150mM NaCl透析。在透析缓冲液中使用搅拌棒。透析管的MWCO应为3.5 kDa。透析体积应足以在完全平衡后获得15mM的最终咪唑浓度
    22. 用2.5 CV 10mM HEPES,pH 7.5,150mM NaCl平衡大小排阻色谱柱(Superdex 75 10/300 GL)。
    23. 使用1ml固体NiNTA(如步骤A13中与ddH 2 O和透析缓冲液一起预洗)在4℃旋转10分钟,使透析的材料转动。
    24. 收集浓缩器(3 kDa MWCO)中的流通。浓缩器应用ddH 2 O运行,并用透析缓冲液冲洗。用3 CV透析缓冲液洗涤NiNTA色谱柱,并在浓缩器中收集该物质。
    25. 离心浓缩器,在约3000℃10分钟,4℃。取出并反转几次以使溶液均质化(或者:上下移动)。重复,直至达到所需体积(<0.5 ml)
    26. 将样品转移到预冷的1.5 ml杯中。在4℃的台式离心机中以16,000 x g离心10分钟。
    27. 将上清液转移到新的预冷杯中
    28. 准备HPLC。将新的管放在馏分收集器中。将注射器分开,用透析缓冲液冲洗。通过快速推出液体从注射器中清除气泡。预先冲洗针头和循环与透析缓冲液。
    29. 将样品注入循环。开始运行一旦峰值接近,将分馏体积改为0.3毫升。峰值变化至1.4ml分馏后
    30. 根据色谱图确定哪些馏分含有峰。使用Nanodrop确定这些级分中的蛋白质浓度。
    31. 池峰分数和浓缩物(3kDa MWCO)至最终浓度超过10mg/ml。
    32. 将样品储存在4°C直至使用。 Nb5776保持稳定至少3个月。

  2. 生产和净化SLC26Dg
    1. 变换E。大肠杆菌MC1061与pBXC3GH_SLC26Dg。在含有100μg/ml氨苄青霉素的LB-琼脂平板上培养。在37°C过夜生长。
    2. 挑一个殖民地接种100 ml TB/Amp的预培养。在37°C过夜生长。
    3. 在发酵罐中接种90升预培养的9L TB/Amp,并在37℃下培养。调节搅拌和曝气以保证快速增长。将容器压力调节至大约1巴超压,以减少发泡,并且如果形成泡沫,则用10%(v/v)聚丙二醇2000的小等分试样补充培养物。继续培养直到达到约2的OD 600。
    4. 在1小时内逐渐将温度降至25°C。另外20分钟后,用2.5ml 20%(w/v)L-阿拉伯糖诱导培养。继续孵化一夜。
    5. 通过在4℃下以5,500×g离心15分钟收获细胞。从这里保持样品冷却(0-4°C)和预冷解决方案和设备预先。
    6. 将细胞重悬于50mM KPi,pH 7.5,150mM NaCl中,至600nm至150-200。
    7. 加入溶菌酶(最终1mg/ml),DNAse I(20μg/ml终浓度)和MgSO 4(最终浓度为1mM)。
    8. 使用Ultra-Turrax T25匀浆器使细胞均质化,例如。
    9. 在搅拌下,在4℃下孵育约60分钟。
    10. 使用高压细胞破坏器(Emulsiflex,预冷)以10 kPsi的三次通过破坏细胞。
    11. 加入PMSF(1mM终浓度)和EDTA(5mM终浓度)
    12. 进行低速旋转离心以除去未破裂的细胞,在4℃下以10,000×g/min进行15分钟。
    13. 将上清液转移到适合超速离心的管道上
    14. 进行高速旋转离心以使膜沉淀,1小时,16,000 x g,4℃。
    15. 确定泡囊的净重。
    16. 使用Potter管将囊泡重悬于50mM KPi,pH7.5,150mM NaCl,10%甘油中〜1g/2ml。
    17. 以1/5/10 g等份的液氮冷冻囊泡。
    18. 将等分试样储存在-80°C直到使用。 1年以上可以储存囊泡
    19. 在室温水和搅拌棒的烧杯中解冻5g膜泡。解冻后,将样品放在冰上。
    20. 将癸基麦芽糖(粉末级)粉末平衡至室温
    21. 将粘稠的囊泡悬浮液转移到50ml管中。用冰冷的50mM KPi,pH7.5,150mM NaCl,10%甘油洗涤管,并将体积调节至40ml。
    22. 加入咪唑至最终浓度为15 mM
    23. 加入0.625g癸基麦芽糖苷,得到约1.6%的终浓度。保持泡沫:洗涤剂比例约为5g:0.5-0.6g是很重要的,以获得良好的增溶作用。
    24. 在4℃下孵育1小时,旋转。
    25. 在4℃下以160,000×g离心该材料30分钟。
    26. 准备NiNTA柱。使用3毫升固体NiNTA。用〜10 CV(30 ml)ddH 2 O和约10 CV(30 ml)50 mM KPi,pH 7.5,150 mM NaCl,10%甘油洗涤柱。
    27. 在4℃下,用NiNTA孵育上清1小时,旋转
    28. 排水柱。
    29. 用20 CV(60ml)20mM HEPES,pH7.5,150mM NaCl,10%甘油,50mM咪唑,0.2%癸基麦芽糖苷洗涤柱。
    30. 洗脱柱,其中0.5份(1.5ml)20mM HEPES,pH7.5,150mM NaCl,10%甘油,300mM咪唑,0.2%癸基麦芽糖苷等分试样。
    31. 使用Nanodrop确定蛋白质浓度
    32. 池峰分数,并加入3C蛋白酶至最终蛋白质:3C蛋白酶摩尔比为5:1。
    33. 在4℃下消化2小时,同时对20mM HEPES,pH 7.5,150mM NaCl,10%甘油,0.13%癸基麦芽糖苷透析。在透析缓冲液中使用搅拌棒。透析管的MWCO应为8 kDa。透析体积应足以在完全平衡后获得15mM的最终咪唑浓度
    34. 用2.5 CV 10mM HEPES,pH 7.5,150mM NaCl,0.2%癸基麦芽糖苷(最高纯度)平衡大小排阻色谱柱(Superdex 200 10/300 GL)。
    35. 使用0.6ml固体NiNTA(如步骤B26)在透析缓冲液中预先洗涤并在4℃下旋转10分钟,使透析的材料孵育。
    36. 在浓缩器(50kDa MWCO)中收集流通。浓缩器应用ddH 2 O运行,并用透析缓冲液冲洗。用3 CV透析缓冲液洗涤NiNTA色谱柱,并在浓缩器中收集此流量。
    37. 离心浓缩器,在3,000 x g,4℃下10分钟。取出并反转几次以使溶液均质化(或者:上下移动)。重复,直至达到所需体积(<0.5 ml)
    38. 将样品转移到预冷的1.5 ml杯中。离心10分钟,在4°C的台式离心机中,16,000 x g
    39. 将上清液转移到新的预冷杯中
    40. 准备HPLC。将新的管放在馏分收集器中。将注射器分开,用透析缓冲液冲洗。通过快速推出液体从注射器中清除气泡。预先冲洗针头和循环与透析缓冲液。
    41. 将样品注入循环。开始运行一旦峰值接近,将分馏体积改为0.3毫升。峰值变化至1.4ml分馏后
    42. 根据色谱图确定哪些馏分含有峰。使用Nanodrop确定这些级分中的蛋白质浓度。
    43. 池峰分数和浓缩物(50kDa MWCO)至最终浓度超过10mg/ml。
    44. 不要存放样品,但立即进行复杂的形成和结晶。

  3. SLC26Dg-Nb5776的络合物形成和结晶
    1. 补充纯化和浓缩的具有癸基麦芽糖苷(10%w/v原料)的Nb5776至0.2%(w/v)的最终浓度。
    2. 以1:2的摩尔比混合纯化的SLC26Dg和Nb5776。在4°C孵育10分钟。
    3. 使用用10mM HEPES,pH 7.5,150mM NaCl,0.2%癸基麦芽糖平衡平衡的Superdex 200 10/300GL柱将样品提交给SEC。
    4. 根据色谱图确定哪些馏分含有峰。使用Nanodrop确定这些级分中的蛋白质浓度。通过SDS-PAGE验证级分中两种蛋白质的存在。
    5. 池峰分数和浓缩物(50kDa MWCO)至最终浓度约9-12mg/ml。
    6. 在4℃下通过蒸气扩散在座滴中生长SLC26Dg-Nb5776晶体(1μl蛋白质+1μl储存溶液[参见食谱])。以1:1的比例混合蛋白质和储层溶液。约1周后出现晶体。

数据分析

代表性数据如图2和3所示

图2.纯化SLC26Dg。根据本文详述的方案,在SLC26Dg纯化期间在不同步骤获得的样品的SDS-PAGE分析。 "洗脱","消化"和"SEC"是指在用HRV 3C蛋白酶消化后和从SEC柱洗脱后,从NiNTA柱洗脱后得到的样品。黑色和白色箭头分别表示含有GFP(与SLC26Dg或单独融合)和SLC26Dg的条带。右图是通过测量凝胶GFP荧光获得的SDS-PAGE凝胶的图像。左图是相同凝胶的考马斯染色图像。左侧显示分子量标记(kDa)。



图3. SLC26Dg和Nb5776的复合物形成A.如方案所示,用过量的Nb5776预温育的SLC26Dg的大小排阻色谱图。 B.来自SEC柱的相关峰分数的SDS-PAGE分析。 C.SLC26Dg-Nb5776复合物的蛋白质晶体。

食谱

  1. LB琼脂(1升)
    10 g try pton pton。。。 5克酵母提取物
    5克NaCl
    15克琼脂
    将软化的H 2 O 2加入1L,并在121℃下高压灭菌20分钟
  2. TB培养基(1升)
    1. 12 g try pton pton。。。。 24克酵母提取物
      8.6 ml 86%(w/v)甘油 将软化的H 2 O 2加入到950ml以上
    2. 50毫升1.44 MK 2 HPO 4,在ddH 2中的0.34M KH 2 PO 4 sub> O
      分别对两种溶液进行高压灭菌(121℃,20分钟),并在使用前混合使用
  3. 1 M KPi,pH 7.5(200 ml)
    制备200ml 1KH 2 HPO 4的溶液和100ml 1M KH 2 PO 4的溶液。 >使用ddH 2 O
    将两种溶液以约4:1的比例混合(K 2 HPO 4:KH 2 PO 4)在恒定搅拌的同时测量pH值
    通过使用碱性(K 2 HPO 4)或酸性(KH 2 PO 4)调节pH值,组件
  4. 水库解决方案
    1M甲酸铵,50mM乙酸钠,pH4.5
    45-50%PEG400(w/v)

致谢

这里详述的协议是在苏黎世大学Raimund Dutzler教授的实验室开发的,受益于Drs的有用建议。 Sandra Markovic,Ricarda J.C. Hilf,Ines Ehrnstorfer,Stefan Warmuth,Iwan Zimmermann和Markus Seeger教授。来自Vrije Universiteit Brussel的Jan Steyaert教授和Els Pardon博士被公认为生产和选择Nb5776。苏黎世大学蛋白质结晶中心的Beat Blattman和CélineStutz-Ducommun表示支持SLC26Dg/Nb5776建立结晶条件。尔格。承认人类边疆科学计划(LT-00899/2008)和德国研究基金会通过法兰克福高分子复合物卓越团队的长期奖学金的支持。

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引用:Chang, Y., Shaik, F. R., Neldner, Y. and Geertsma, E. R. (2017). Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies. Bio-protocol 7(3): e2116. DOI: 10.21769/BioProtoc.2116.
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