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Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)
重组拟南芥单糖半酰基二酰基甘油合酶(MGD1)的表达、纯化和结晶   

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Abstract

In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant form of Arabidopsis thaliana (A. thaliana) monogalactosyldiacylglycerol synthase 1 (MGD1) protein, in Escherichia coli (E. coli), using a two-step chromatographic purification procedure. The protein is easily expressed and purified to milligram quantities, suitable for biochemical and structural studies. The crystallization of MGD1 is also described.

Keywords: Photosynthetic tissues(光合组织), Galactolipids(半乳糖脂), Monogalactosyldiacylglycerol(单半乳糖基二酰基甘油), Crystallization(结晶), MGDG synthase(MGDG合酶)

Background

Previous attempts to express plant MGDs in E. coli showed that approximately 99% of the recombinant protein accumulated in inclusion bodies (Miège et al., 1999). Solubilization of bacterial membranes using detergents, or in vitro inclusion bodies refolding protocols were developed and yielded pure and active fractions, sufficient to monitor the activity of the enzyme, but not to pursue its structural study (Nishiyama et al., 2003; Botté et al., 2005). Using a combination of different biochemical and biophysical techniques, and investigating the effects of various buffers and additives on the biochemical behavior of the enzyme, a simple, efficient and fast protocol was developed for the expression and purification of recombinant MGD1, addressing the problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation (Rocha et al., 2013). Conditions detailed here allowed the unprecedented production of a pure, soluble and active form of MGD1 and comply with both structural and functional dissections of this enzyme (Rocha et al., 2016). The protocol here described can also serve as a starting strategy to purify similar proteins.

Materials and Reagents

  1. 50 ml Corning tubes (Corning, catalog number: 430829 )
  2. 1.5 ml Eppendorf tubes (Treff, catalog number: 96.07246.9.01 )
  3. 30 ml centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3138-0030 )
  4. 0.2 µm PES-membrane filters (Dominique Dutscher, catalog number: 051732 )
  5. Vivaspin 20, MWCO 30,000 Da concentrators (Sartorius, catalog number: VS2022 )
  6. HisTrap FF 1 ml column (GE Healthcare, catalog number: 17-5319-01 )
  7. Superdex S200 10/300 GL column (GE Healthcare, catalog number: 17-5175-01 )
  8. 24-well VDX plate with sealant (HAMPTON RESEARCH, catalog number: HR3-170 )
  9. 22 mm cover slips (HAMPTON RESEARCH, catalog number: HR3-233 )
  10. E. coli BL21(DE3) strain (New England Biolabs, catalog number: C2527I )
  11. Luria Broth (LB) media (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12780-052 )
  12. Kanamycin (Euromedex, catalog number: EU0420 )
  13. Isopropyl-β-D-thiogalactopyranoside (IPTG) (Euromedex, catalog number: EU0008 )
  14. Antifoam 204 (Sigma-Aldrich, catalog number: A6426 )
  15. Benzonase® nuclease (Sigma-Aldrich, catalog number: E1014
  16. Imidazole (Sigma-Aldrich, catalog number: 56749 )
  17. Ethanol absolute anhydrous (CARLO ERBA Reagents, catalog number: 308607 )
  18. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
  19. PEG 3350 (Sigma-Aldrich, catalog number: 88276 )
  20. Liquid nitrogen (LN2)
  21. Glycerol (Sigma-Aldrich, catalog number: G7757 )
  22. HEPES (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP310 )
  23. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10553515 )
  24. Urea (Sigma-Aldrich, catalog number: U1250 )
  25. Dithiothreitol (DTT) (Euromedex, catalog number: EU0006 )
  26. cOmpleteTM protease inhibitor cocktail (Roche Diagnostics, catalog number: 11873580001 )
  27. Bis-tris propane (Sigma-Aldrich, catalog number: B6755 )
  28. Tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich, catalog number: C4706 )
  29. Tris base (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10376743 )
  30. Sodium dodecyl sulfate (SDS) 10% (Euromedex, catalog number: EU0760 )
  31. Bromophenol blue (EMD Millipore, catalog number: 108122 )
  32. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  33. Hydrochloric acid 37% (CARLO ERBA Reagents, catalog number: 403871 )
  34. Sodium hydroxide (EMD Millipore, catalog number: 1064621000 )
  35. Lysis buffer (see Recipes)
  36. Washing/binding buffer (see Recipes)
  37. Elution buffer (see Recipes)
  38. Size exclusion buffer (see Recipes)
  39. SDS-PAGE deposition buffer (5x DB) (see Recipes)
  40. 20% (v/v) ethanol (see Recipes)
  41. 6 N hydrochloric acid solution (for pH adjustment) (see Recipes)
  42. 10 M sodium hydroxide solution (for pH adjustment) (see Recipes)
  43. Precipitant solution (or mother liquor) (see Recipes)

Equipment

  1. Culture flasks (IDEA Conception, catalog number: ART0004104 )
    Note: These are custom made 3 L culture flasks.
  2. Orbital shakers at 30 °C and 37 °C
  3. Incubator at 20 °C (for crystallization plates)
  4. Sonication bath (room temperature)
  5. Lab balances
  6. Cooled centrifuges
  7. Filtration system or syringes
  8. Cell Disruptor CSL ‘One-shot’ (Constant Systems, Ltd)
  9. ÄKTAFPLC instrument (GE Healthcare, catalog number: 18190026 ) or equivalent
  10. SDS-PAGE equipment
  11. Zetasizer Nano ZSTM for Dynamic Light Scattering Analysis (Malvern Instruments, model: ZEN3600 ) or equivalent
  12. BioPhotometer 6131 (Eppendorf, Germany)
  13. NanoDropTM ND-2000 spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: ND-2000 )
  14. Olympus Zoom Stereo microscope (Olympus, model: SZ1145 TR CTV ) or equivalent
    Note: This product has been discontinued.
  15. Olympus Transmitted Light Illumination Base (Olympus, model: SZX-ILLK200 ) or equivalent
    Note: This product has been discontinued.

Note: Cryocrystallography material for crystals manipulation in liquid nitrogen (LN2)

  1. Litholoops several sizes (Molecular Dimensions)
  2. CryoCaps (Plain) (Molecular Dimensions, catalog number: MD7-400 )
  3. Magnetic CryoVials (Molecular Dimensions, catalog number: MD7-402 )
  4. EMBL/ESRF Sample changer starter kit (Molecular Dimensions, catalog number: MD7-500 )
  5. Magnetic Cryo Wand (Molecular Dimensions, catalog number: MD7-411 )
  6. LN2 foam dewars (Molecular Dimensions, catalog number: MD7-35 )
  7. Dry shipper (CX100) dewar (Molecular Dimensions, catalog number: MD7-21 )

Procedure

  1. Expression of MGD1△137-6His protein
    The protein construction here described is a truncated form of MGD1 (MGD1△137-6His), deprived of its N-terminal 136 amino-acid residues and with a non-cleavable 6-histidine tag at the protein C-terminus. The deleted segment comprises the canonical N-terminal chloroplast signal peptide (res. 1-106; cleaved upon import to the organelle), and a 30-mer region predicted to be disordered, and not necessary for activity. The mgd1137 DNA fragment was cloned at the NdeI/NotI sites of the pET29b vector (Novagen, 69872), generating the pET29b-MGD1△137-6His construct. E. coli BL21 (DE3) cells were used as host cells for protein production.
    1. Prepare 1 L of sterile LB medium supplemented with kanamycin (30 µg/ml). Inoculate media with E. coli BL21 (DE3) cells harboring the pET29b-MGD1△137-6His construct. Incubate culture at 37 °C and 180 rpm.
    2. Monitor OD600nm of culture on a UV/VIS spectrophotometer.
    3. At OD600nm ~0.6-0.8, take 1 ml sample of culture for SDS-PAGE analysis (before induction - T0). Centrifuge (13,000 x g, 1 min), discard supernatant and resuspend pellet in 40 µl of distilled water and 10 µl of 5x DB. Boil samples for 5 min and sonicate for 2 min prior to gel loading.
    4. Induce cells with IPTG to a final concentration of 0.25 mM. Incubate 4 h at 30 °C, 180 rpm.
    5. After induction, take 1 ml sample of culture for SDS-PAGE analysis (after induction - T4). Centrifuge (13,000 x g, 1 min), discard supernatant and resuspend pellet in 80 µl of distilled water and 20 µl of 5x DB. Boil samples for 5 min and sonicate for 2 min prior to gel loading.
    6. Harvest cells by centrifugation (7,000 x g for 20 min at 4 °C).
    7. Discard supernatant and store the pellet in a previously weighed 50 ml Corning tube.
    8. Weigh wet cell pellet and store at -20 °C if not immediately used (typically ~4 g/L culture).

  2. IMAC purification of MGD1△137-6His protein
    1. Resuspend cell pellet in the lysis buffer (typically 5 ml/g of cells). If cell resuspension is difficult, up to 10 ml/g of cells of lysis buffer can be used.
    2. Add Antifoam 204 (1-2 drops for ~20 ml).
    3. Disrupt cells on a Cell Disruption system at 1.5-1.8 kbar and incubate lysate for 20 min on ice. If lysate is too viscous upon disruption, add more Benzonase nuclease and/or increase incubation time on ice.
    4. Spin lysate at 45,000 x g for 30 min at 4 °C. Recover supernatant, dilute 2x in binding/washing buffer and further clarify it by filtration using a 0.2 µm filter.
    5. Equilibrate the Histrap FF 1 ml (or 5 ml for scale-up) with 5-10 CV (column volumes) of binding/washing buffer at 1 ml/min (2 ml/min on the 5 ml column).
    6. Inject clarified lysate in the column at 1 ml/min. Collect the column flow-through.
    7. Wash column with the same buffer until Abs280nm is stable.
    8. Wash column at 1 ml/min (2 ml/min on the 5 ml column) with 5-10 CV of 8% elution buffer (40 mM imidazole) followed by 5-10 CV of 16% (80 mM imidazole) of the same solution.
    9. Elute proteins at 1 ml/min (2 ml/min for the 5 ml column) using a linear gradient from 16-100% of the same buffer (80-500 mM imidazole). Typically, gradient is performed for 10 min (15 min on the 5 ml column).
    10. Collect fractions (1 ml) throughout all purification steps.
    11. Evaluate protein purity of MGD1137-6His containing fractions by SDS-PAGE analysis - take 16 µl of sample and add 4 µl of 5x DB. Boil samples for 5 min and sonicate for 2 min prior to gel loading.
    12. Evaluate protein homogeneity of MGD1137-6His containing fractions by dynamic light scattering (DLS) analysis (see Note 7 for details).
    13. Pool appropriate fractions and concentrate up to 20 mg/ml using a Vivaspin 20, MWCO 30,000 Da concentrator (see Note 8 for details).
    14. Monitor protein concentration by UV absorbance at 280 nm on a NanoDropTM 2000 spectrophotometer, using a sequence-derived extinction coefficient of 47,900 M-1 cm-1. Use 1:1 washing/elution buffer mix [(imidazole)final ~250 mM] as blank.

  3. Size exclusion purification of MGD1△137-6His protein
    1. Equilibrate the Superdex S200 10/300 GL column with 2 CV (48 ml) of size exclusion buffer at 0.5-0.8 ml/min (control that pressure does not exceed the maximum limit of 1.5 MPa, otherwise decrease the flow).
    2. Inject a maximum of 500 µl of a 20 mg/ml protein sample in the column.
    3. Wash the column with the same buffer to elute proteins.
    4. Collect 0.5 ml fractions and perform SDS-PAGE and DLS analysis of the samples. For the SDS-PAGE, take 16 µl of sample and add 4 µl of 5x DB. Boil samples for 5 min and sonicate for 2 min prior to gel loading. If samples are too viscous for gel loading, increase boiling and/or sonication times.
    5. Wash the column with 2 CV of Mili-Q (MQ) water, followed by 2 CV of 20% ethanol.
    6. Pool fractions containing pure and homogeneous MGD1△137-6His protein.
    7. Concentrate the protein to ~10 mg/ml using a Vivaspin 20, MWCO 30,000 concentrator.
    8. Monitor protein concentration by UV absorbance at 280 nm, using the size exclusion buffer as blank.
    9. Prepare 40 µl aliquots and store at -20 °C, until further use.

  4. Crystallization of MGD1△137-6His protein
    1. Prepare a protein stock solution at 6 mg/ml (use the size exclusion buffer for dilution).
    2. Prepare the precipitant solution containing 200 mM MgCl2·6H2O and 22% (w/v) PEG 3350.
    3. Pipet 400 µl of precipitant solution into the reservoir(s) of the crystallization plate.
    4. Prepare the crystallization drop(s) on the cover slip with 1 µl of MGD1137-6His at 6 mg/ml and 2 µl of precipitant solution.
    5. Place the cover slip over the well (drop facing reservoir) and seal.
    6. Incubate plate at 20 °C. Inspect the plate frequently, under a microscope (crystals appear in one week, but often grow bigger within 10-12 days). Avoid temperature changes and keep the plate at 20 °C as much as possible.
    7. Crystals cryoprotection is achieved in a two-step procedure (i) by transferring into a stabilizing solution of mother liquor (precipitant solution) containing 27% (w/v) PEG 3350 precipitant, (ii) followed by a quick soak in the same cryoprotective solution supplemented with 30% (w/v) of the same precipitant. Use the cryocrystallography material to mount and handle crystals.
    8. Flash-cool and store crystals in LN2.

Notes

  1. A more detailed explanation on the role of MGD1 in galactolipid biosynthesis is described in Rocha et al. (2013; 2016).
  2. All solutions must be filtered prior to use. Solutions to be used in size exclusion chromatography have also to be degassed.
  3. To accelerate cell growth, a pre-inoculum can be used. Prepare a small culture using the procedure described, and incubate overnight at 37 °C, 180 rpm. Inoculate the new LB media with 2-3% of the overnight culture (typically 20-30 ml/L of media).
  4. Length of column washing/equilibration steps can be adjusted (shortened or extended), by monitoring the stabilization of the absorbance at 280 nm, as given by the AKTA FPLC system.
  5. Because MGD1137-6His has a molecular weight of 45 kDa, 10% acrylamide SDS-PAGE gels can be used.
  6. DTT can be used during the procedure, but for long-term storage TCEP is more stable and efficient.
  7. DLS is an ideal technique for characterizing proteins in a variety of conditions and obtain information on their state in solution. It can be used to identify conditions that provide biologically relevant oligomeric structures, ensure monodispersity and optimal stability, minimize aggregation and ultimately, are ideal for crystallization. The default standard operation protocol (SOP) for protein size analysis of the Zetasizer Nano ZSTM was used, with modifications to temperature (4 °C or 20 °C) and type of solvent used (e.g., water + 5% glycerol) to take into account the viscosity of the solution. Triplicate measurements are performed automatically. If the sample is monodisperse, only one population is seen (single peak), whereas polydispersity is represented by multiple populations (several peaks); also, the size distribution (peak width) around the protein diameter should be as narrow as possible; and finally the particle diameter should be compatible with the protein species (very large diameters indicate protein aggregation). A typical DLS experiment for MGD1, together with its expected results, can be found in Rocha et al. (2013), where the protein production optimized protocol was first published.
  8. Throughout the purification, the appropriate fractions are determined according to their purity (SDS-PAGE) and homogeneity (DLS). MGD1137-6His fractions that are not very pure or not homogeneous are discarded from the pooling and not used in the following steps. Typically, fractions from the beginning and end of the eluted chromatographic peak are not included in the pooling.
  9. Several tutorials are available showing how to crystallize a protein in vapour diffusion techniques. Some useful links to tutorials are given here:
    1. xray.bmc.uu.se/terese/tutorials.html – A tutorial by Terese Bergfors on protein crystallization
    2. https://www.youtube.com/watch?v=gLsC4wlrR2A – Understanding Crystallography, Part 1: From proteins to crystals
    3. https://www.youtube.com/watch?v=Grh8-pHpAJ4 – Protein crystallization hanging drop vapour diffusion

Recipes

  1. Lysis buffer
    25 mM HEPES buffer
    500 mM NaCl
    1 M urea
    10 mM imidazole
    1 mM DTT
    Adjust pH to 7.5
    Add Benzonase® nuclease (1 µl/10 ml of solution) and cOmpleteTM protease inhibitor cocktail (1 tablet/50 ml solution)
  2. Washing/binding buffer
    25 mM HEPES
    500 mM NaCl
    1 M urea
    10 mM imidazole
    1 mM DTT
    Adjust pH to 7.5
  3. Elution buffer
    25 mM HEPES
    500 mM NaCl
    1 M urea
    500 mM imidazole
    1 mM DTT
    Adjust pH to 7.5
  4. Size exclusion buffer
    25 mM Bis-Tris propane
    150 mM NaCl
    5% (v/v) glycerol
    1 mM TCEP
    Adjust to pH 6.5
  5. SDS-PAGE deposition buffer (5x DB)
    250 mM Tris, pH 6.8
    50 % (v/v) glycerol
    10 % (v/v) SDS
    0.05% (w/v) bromophenol blue
    250 mM DTT
  6. 20% (v/v) ethanol
  7. 6 N hydrochloric acid solution (for pH adjustment)
  8. 10 M sodium hydroxide solution (for pH adjustment)
  9. Precipitant solution (or mother liquor)
    200 mM MgCl2·6H2O
    22% (w/v) PEG 3350

Acknowledgments

This protocol is an extension of that described in Rocha et al., 2013 and Rocha et al., 2016. This work was supported by Agence Nationale de la Recherche (grant ANR- 10-BLAN-1524, ReGal) and by the Rhône-Alpes region (France).

References

  1. Botté, C., Jeanneau, C., Snajdrova, L., Bastien, O., Imberty, A., Breton, C. and Marechal, E. (2005). Molecular modeling and site-directed mutagenesis of plant chloroplast monogalactosyldiacylglycerol synthase reveal critical residues for activity. J Biol Chem 280(41): 34691-34701.
  2. Miège, C., Maréchal, E., Shimojima, M., Awai, K., Block, M. A., Ohta, H., Takamiya, K., Douce, R. and Joyard, J. (1999). Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG. Eur J Biochem 265(3): 990-1001.
  3. Nishiyama, Y., Hardré-Liénard, H., Miras, S., Miège, C., Block, M. A., Revah, F., Joyard, J. and Marechal, E. (2003). Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants. Protein Expr Purif 31(1): 79-87.
  4. Rocha, J., Audry, M., Pesce, G., Chazalet, V., Block, M. A., Maréchal, E. and Breton, C. (2013). Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies. Biochimie 95(4): 700-708.
  5. Rocha, J., Sarkis, J., Thomas, A., Pitou, L., Radzimanowski, J., Audry, M., Chazalet, V., de Sanctis, D., Palcic, M. M., Block, M. A., Girard-Egrot, A., Marechal, E. and Breton, C. (2016). Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants. Plant J 85(5): 622-633. 

简介

在植物细胞中,半乳糖脂是主要的,代表高达光合组织脂质含量的50%。通过使用UDP-半乳糖作为供体糖和二酰基甘油(DAG)作为受体的MGDG合成酶(MGD)启动半乳糖脂合成以形成单糖基二酰基甘油(MGDG)。该方案用于在大肠杆菌中产生拟南芥(Arabidopsis thaliana)(拟南芥)单糖半乳糖二酰基甘油合酶1(MGD1)蛋白的重组形式(大肠杆菌),使用两步色谱纯化方法。蛋白质容易表达和纯化至数量,适用于生物化学和结构研究。还描述了MGD1的结晶。

背景 以前在e中表达植物MGD的尝试。大肠杆菌显示约99%的重组蛋白质积累在包涵体中(Miège等,1999)。开发了使用洗涤剂或体外包涵体折叠方案的细菌膜的溶解,并产生足够的纯和活性级分,足以监测酶的活性,但不进行其结构研究(Nishiyama et al。,2003;Botté等人,2005)。利用不同的生物化学和生物物理技术的组合,并研究了各种缓冲液和添加剂对酶的生物化学行为的影响,开发了一种简单,有效和快速的方案用于重组MGD1的表达和纯化,解决了经常遇到的问题通过糖基转移酶的纯化,特别是蛋白质聚集(Rocha等人,2013)。这里详述的条件允许前所未有地生产纯的,可溶性和活性形式的MGD1,并且符合该酶的结构和功能性解剖(Rocha等人,2016)。这里描述的方案也可以作为纯化相似蛋白质的起始策略。

关键字:光合组织, 半乳糖脂, 单半乳糖基二酰基甘油, 结晶, MGDG合酶

材料和试剂

  1. 50ml康宁管(Corning,目录号:430829)
  2. 1.5ml Eppendorf管(Treff,目录号:96.07246.9.01)
  3. 30ml离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3138-0030)
  4. 0.2μmPES膜过滤器(Dominique Dutscher,目录号:051732)
  5. Vivaspin 20,MWCO 30,000 Da集中器(Sartorius,目录号:VS2022)
  6. HisTrap FF 1 ml柱(GE Healthcare,目录号:17-5319-01)
  7. Superdex S200 10/300 GL柱(GE Healthcare,目录号:17-5175-01)
  8. 带密封剂的24孔VDX板(HAMPTON RESEARCH,目录号:HR3-170)
  9. 22毫米封面(HAMPTON RESEARCH,目录号:HR3-233)
  10. E。大肠杆菌BL21(DE3)菌株(New England Biolabs,目录号:C2527I)
  11. Luria Broth(LB)培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:12780-052)
  12. 卡那霉素(Euromedex,目录号:EU0420)
  13. 异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(Euromedex,目录号:EU0008)
  14. 消泡剂204(Sigma-Aldrich,目录号:A6426)
  15. 苯佐酶核酸酶(Sigma-Aldrich,目录号:E1014) 
  16. 咪唑(Sigma-Aldrich,目录号:56749)
  17. 乙醇无水无水(CARLO ERBA试剂,目录号:308607)
  18. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M9272)
  19. PEG 3350(Sigma-Aldrich,目录号:88276)
  20. 液氮(LN 2 2)
  21. 甘油(Sigma-Aldrich,目录号:G7757)
  22. HEPES(Thermo Fisher Scientific,Fisher Scientific,目录号:BP310)
  23. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:10553515)
  24. 尿素(Sigma-Aldrich,目录号:U1250)
  25. 二硫苏糖醇(DTT)(Euromedex,目录号:EU0006)
  26. cOmplete TM 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11873580001)
  27. 双三丙烷(Sigma-Aldrich,目录号:B6755)
  28. 三(2-羧乙基)膦(TCEP)(Sigma-Aldrich,目录号:C4706)
  29. Tris基(Thermo Fisher Scientific,Fisher Scientific,目录号:10376743)
  30. 十二烷基硫酸钠(SDS)10%(Euromedex,目录号:EU0760)
  31. 溴酚蓝(EMD Millipore,目录号:108122)
  32. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  33. 盐酸37%(CARLO ERBA试剂,目录号:403871)
  34. 氢氧化钠(EMD Millipore,目录号:1064621000)
  35. 裂解缓冲液(见配方)
  36. 洗涤/结合缓冲液(见配方)
  37. 洗脱缓冲液(见配方)
  38. 大小排除缓冲区(见配方)
  39. SDS-PAGE沉积缓冲液(5x DB)(参见食谱)
  40. 20%(v / v)乙醇(参见食谱)
  41. 6 N盐酸溶液(用于pH调节)(见配方)
  42. 10 M氢氧化钠溶液(用于pH调节)(见配方)
  43. 沉淀液(或母液)(见配方)

设备

  1. 文化瓶(IDEA构想,目录号:ART0004104)
    注意:这些是定制的3升培养瓶。
  2. 轨道震荡器在30°C和37°C
  3. 孵化器在20°C(用于结晶板)
  4. 超声波浴(室温)
  5. 实验室平衡
  6. 冷却离心机
  7. 过滤系统或注射器
  8. 细胞破坏者CSL"一次性"(Constant Systems,Ltd)
  9. ÄKTAFPLC仪器(GE Healthcare,目录号:18190026)或等效物
  10. SDS-PAGE设备
  11. Zetasizer Nano ZS TM 用于动态光散射分析(Malvern Instruments,型号:ZEN3600)或等效物
  12. BioPhotometer 6131(德国Eppendorf)
  13. NanoDrop TM ND-2000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:ND-2000)
  14. 奥林巴斯Zoom立体显微镜(Olympus,型号:SZ1145 TR CTV)或等效物
    注意:本产品已停产。
  15. 奥林巴斯透射灯照明基地(Olympus,型号:SZX-ILLK200)或等效物
    注意:本产品已停产。

注意:用于在液氮中操作晶体的晶体析出材料(LN 2/2)

  1. Litholoops几种尺寸(分子尺寸)
  2. CryoCaps(Plain)(分子尺寸,目录号:MD7-400)
  3. 磁性CryoVials(分子尺寸,目录号:MD7-402)
  4. EMBL / ESRF样品更换器入门套件(分子尺寸,目录号:MD7-500)
  5. 磁性冷冻棒(分子尺寸,目录号:MD7-411)
  6. 泡沫杜瓦瓶(分子尺寸,目录号:MD7-35)
  7. 干托运人(CX100)杜瓦瓶(分子尺寸,目录号:MD7-21)

程序

  1. MGD1的表达△137-6His蛋白质
    这里描述的蛋白质构建是一种截短形式的MGD1(MGD1△137-6His),其蛋白C末端被剥夺了其N-末端136个氨基酸残基和不可裂解的6-组氨酸标签。缺失的片段包含典型的N-末端叶绿体信号肽(重排1-106;在进入细胞器时被切割)和预测为无序的30聚体区,并不是活性所必需的。 DNA片段已被克隆在我的Ide I / I pET29b载体的位点(Novagen,69872),产生pET29b-MGD1△137-6His构建体。 E。大肠杆菌BL21(DE3)细胞用作蛋白质生产的宿主细胞。
    1. 准备1L补充有卡那霉素(30μg/ ml)的无菌LB培养基。接受媒体与 E。含有pET29b-MGD1△137-6His构建体的大肠杆菌BL21(DE3)细胞。在37℃和180rpm下孵育培养物。
    2. 在UV / VIS分光光度计上监测培养物的OD 600nm。
    3. 在OD 600nm〜0.6-0.8,取1ml培养物用于SDS-PAGE分析(诱导前T0)。离心(13,000×g,1分钟),弃去上清并将沉淀重悬于40μl蒸馏水和10μl5×DB中。煮沸样品5分钟,并在凝胶加载之前超声处理2分钟
    4. 用IPTG诱导细胞至0.25mM的终浓度。在30℃,180rpm下孵育4小时。
    5. 诱导后,取1 ml培养液进行SDS-PAGE分析(诱导后T4)。离心机(13,000×g,1分钟),弃去上清并将沉淀重悬于80μl蒸馏水和20μl5×DB中。煮沸样品5分钟,并在凝胶加载之前超声处理2分钟
    6. 通过离心收集细胞(7,000 x g在4℃下20分钟)
    7. 丢弃上清液并将颗粒储存在以前称重的50ml科林管中
    8. 称重湿细胞沉淀并储存在-20°C,如果不立即使用(通常约4 g / L培养)
  2. IMAC纯化MGD1△137-6His蛋白质
    1. 将细胞沉淀重悬于裂解缓冲液(通常为5ml / g细胞)。如果细胞再悬浮是困难的,可以使用高达10ml / g的裂解缓冲液细胞。
    2. 加入消泡剂204(1-2滴〜约20毫升)
    3. 在1.5-1.8 kbar的细胞破坏系统上破坏细胞,并在冰上孵育裂解液20分钟。如果裂解物在破裂时粘度过大,可加入更多的Benzonase核酸酶和/或在冰上增加孵化时间。
    4. 在4℃下以45,000×g旋转裂解物30分钟。恢复上清液,在结合/洗涤缓冲液中稀释2x,并通过使用0.2μm过滤器过滤进一步澄清
    5. 用5-10毫升(柱体积)的结合/洗涤缓冲液,以1毫升/分钟(5毫升柱为2毫升/分钟)平衡1分钟(或5毫升放大)。
    6. 在柱中以1ml / min注入澄清的裂解物。收集列流通。
    7. 用相同的缓冲液洗涤柱直到Abs <280nm 是稳定的
    8. 以5-10毫升8%洗脱缓冲液(40毫摩尔咪唑)洗涤1毫升/分钟(5毫升柱2毫升/分钟),然后加5-10微克16%(80毫摩尔咪唑)相同解。
    9. 使用16-100%相同缓冲液(80-500mM咪唑)的线性梯度洗脱1ml / min的洗脱蛋白(5ml柱的2ml / min)。通常,梯度进行10分钟(在5ml柱上15分钟)
    10. 在所有纯化步骤中收集级分(1ml)
    11. 评估MGD1的蛋白质纯度 / span> 137-6通过SDS-PAGE分析含有级分 - 取16μl样品,加入4μl5x DB。煮沸样品5分钟,并在凝胶加载之前超声处理2分钟
    12. 评估MGD1的蛋白质同质性△< / span> 137-6通过动态光散射(DLS)分析包含分数(详见附注7)。
    13. 使用Vivaspin 20,MWCO 30,000 Da浓缩器(参见注释8)了解适当的级分并浓缩至20 mg / ml。
    14. 在NanoDrop TM 2000分光光度计上使用序列衍生的消光系数为47,900M -1,在280nm下通过紫外吸收来监测蛋白质浓度 sup>。使用1:1洗涤/洗脱缓冲液混合物[(咪唑)最终<250>]作为空白。

  3. 大小排除纯化MGD1△137-6His蛋白
    1. 将Superdex S200 10/300 GL柱与2 CV(48ml)的排阻缓冲液以0.5-0.8ml / min(控制压力不超过1.5MPa的最大限度,否则减少流量)平衡。 >
    2. 在柱中注入最多500μl的20 mg / ml蛋白质样品。
    3. 用相同的缓冲液洗涤柱子以洗脱蛋白质。
    4. 收集0.5ml级分,并对样品进行SDS-PAGE和DLS分析。对于SDS-PAGE,取16μl样品,加入4μl5x DB。煮沸样品5分钟,并在凝胶加载之前超声处理2分钟。如果样品太粘稠,无法凝胶装载,则会增加煮沸和/或超声时间
    5. 用2 CV的Mili-Q(MQ)水洗涤柱子,然后用2 CV的20%乙醇洗涤
    6. 含有纯均匀的MGD1△137-6His蛋白的泳池级分。
    7. 使用Vivaspin 20,MWCO 30,000浓缩器将蛋白质浓缩至约10mg / ml
    8. 使用大小排阻缓冲液作为空白,在280nm处通过紫外吸收来监测蛋白质浓度
    9. 准备40μl等分试样并储存于-20°C,直至进一步使用。

  4. 结晶MGD1△137-6His蛋白质
    1. 准备6毫克/毫升的蛋白质储备溶液(使用大小排阻缓冲液进行稀释)
    2. 制备含有200mM MgCl 2·6H 2 O和22%(w / v)PEG 3350的沉淀溶液。
    3. 将400μl沉淀剂溶液吸入结晶板的储存器中。
    4. 使用1μlMGD1准备结晶滴(1)MGD1 137-6为6 mg / ml和2μl沉淀剂溶液。
    5. 将盖板放在井上(面向水箱)并密封。
    6. 孵育板在20°C。经常在显微镜下检查板(晶体出现在一周内,但常常在10-12天内变大)。避免温度变化,尽可能使板材保持在20°C
    7. 晶体冷冻保护是通过转移到含有27%(w / v)PEG 3350沉淀剂的母液(沉淀剂溶液)的稳定溶液中(ii),然后在相同的冷冻保护剂中快速浸泡补充有30%(w / v)相同沉淀剂的溶液。使用微晶玻璃材料来安装和处理晶体。
    8. 闪光冷却并存储在LN <2>中的晶体。

笔记

  1. 关于MGD1在半乳糖脂生物合成中的作用的更详细的解释描述于Rocha等人。 (2013; 2016)。
  2. 所有解决方案必须在使用前进行过滤。用于尺寸排阻色谱的溶液也要脱气
  3. 通过监测由AKTA FPLC系统给出的280nm处的吸光度的稳定性,可以调整(缩短或延长)柱洗涤/平衡步骤的长度。
  4. 因为MGD1 137-6H具有45kDa的分子量,可以使用10%丙烯酰胺SDS-PAGE凝胶。
  5. 在过程中可以使用DTT,但对于长期存储,TCEP更加稳定和高效
  6. DLS是在各种条件下表征蛋白质的理想技术,并获得关于其在溶液中的状态的信息。它可用于鉴定提供生物相关寡聚结构的条件,确保单分散性和最佳稳定性,最小化聚集,最终是结晶的理想选择。使用Zetasizer Nano ZS TM蛋白质尺寸分析的默认标准操作方案(SOP),修改温度(4°C或20°C)和使用的溶剂类型(<例如,水+ 5%甘油)以考虑溶液的粘度。自动执行一式三份的测量。如果样品是单分散的,则仅看到一个群体(单峰),而多聚体由多个群体(几个峰)表示;蛋白质直径周围的尺寸分布(峰宽)也应尽可能窄;最后粒径应与蛋白质物质相适应(非常大的直径表示蛋白质聚集)。 MGD1的典型DLS实验及其预期结果可以在Rocha等人中找到。 (2013),其中蛋白质生产优化方案首次发布
  7. 在整个纯化过程中,根据其纯度(SDS-PAGE)和均匀性(DLS)测定合适的级分。 MGD1 137 -6不是非常纯的或不均匀的部分从合并中丢弃,不用于以下步骤。通常,洗脱色谱峰的开始和结束部分不包括在合并中。
  8. 有几个教程可用于显示如何在蒸气扩散技术中结晶蛋白质。这里给出了一些教程的有用链接:
    1. xray.bmc.uu.se/terese/tutorials.html < / a> - Terese Bergfors关于蛋白质结晶的教程
    2. https://www.youtube.com/watch?v=gLsC4wlrR2A < / a> - 了解晶体学,第1部分:从蛋白质到晶体
    3. https://www.youtube.com/watch?v= Grh8-pHpAJ4 - 蛋白质结晶悬滴蒸气扩散

食谱

  1. 裂解缓冲液
    25 mM HEPES缓冲液
    500 mM NaCl
    1 M尿素
    10 mM咪唑
    1 mM DTT
    将pH调节至7.5
    加入Benzonase(1μl/ 10ml溶液)和cOmplete TM蛋白酶抑制剂混合物(1片/ 50ml溶液)。
  2. 洗涤/结合缓冲液
    25 mM HEPES
    500 mM NaCl
    1 M尿素
    10 mM咪唑
    1 mM DTT
    将pH调节至7.5
  3. 洗脱缓冲液
    25 mM HEPES
    500 mM NaCl
    1 M尿素
    500毫克咪唑
    1 mM DTT
    将pH调节至7.5
  4. 大小排除缓冲区
    25mM Bis-Tris丙烷
    150 mM NaCl
    5%(v / v)甘油 1 mM TCEP
    调整至pH 6.5
  5. SDS-PAGE沉积缓冲液(5x DB)
    250 mM Tris,pH 6.8
    50%(v / v)甘油 10%(v / v)SDS
    0.05%(w / v)溴酚蓝
    250 mM DTT
  6. 20%(v / v)乙醇
  7. 6 N盐酸溶液(用于pH调节)
  8. 10M氢氧化钠溶液(用于pH调节)
  9. 沉淀液(或母液)
    200mM MgCl 2·6H 2 O
    22%(w / v)PEG 3350

致谢

该协议是Rocha等人,2013年和Rocha等人,2016年所描述的这一协议的延伸。这项工作得到了国民经济发展局(National National de la Recherche)(授予ANR - 10-BLAN-1524,ReGal)和罗纳 - 阿尔卑斯地区(法国)。

参考文献

  1. Botté,C.,Jeanneau,C.,Snajdrova,L.,Bastien,O.,Imberty,A.,Breton,C.and Marechal,E.(2005)。&lt; a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16009708"target ="_ blank">植物叶绿体单糖基二酰基甘油合成酶的分子建模和定点诱变揭示了活性的关键残留物。 > J Biol Chem 280(41):34691-34701。
  2. Miaege,C.,Maréchal,E.,Shimojima,M.,Awai,K.,Block,MA,Ohta,H.,Takamiya,K.,Douce,R.and Joyard,J。(1999) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/10518794"target ="_ blank"> A型MGDG合酶的生化和拓扑性质,菠菜叶绿体包膜酶催化原核和真核MGDG的合成。 Eur J Biochem 265(3):990-1001。
  3. Nishiyama,Y.,Hardré-Liénard,H.,Miras,S.,Miège,C.,Block,MA,Revah,F.,Joyard,J.and Marechal,E。(2003)。从变性包涵体折叠,纯化至同质性,并简化了来自陆地植物的MGDG合酶测定。蛋白质清除31(1):79-87。
  4. Rocha,J.,Audry,M.,Pesce,G.,Chazalet,V.,Block,MA,Maréchal,E.and Breton,C。(2013)。重新拟南芥中主要的半乳糖脂肪酸合成酶MGD1的表达和纯化,以建立生化和结构研究的新标准。 Biochimie 95(4):700-708。
  5. Rocha,J.,Sarkis,J.,Thomas,A.,Pitou,L.,Radzimanowski,J.,Audry,M.,Chazalet,V.,de Sanctis,D.,Palcic,MM,Block,MA,Girard -Egrot,A.,Marechal,E.和Breton,C。(2016)。&nbsp; MGD1(植物中主要的半乳糖脂合成酶)的结构见解和膜结合特性。植物J 85(5):622-633。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rocha, J., Chazalet, V. and Breton, C. (2016). Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1). Bio-protocol 6(24): e2064. DOI: 10.21769/BioProtoc.2064.
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