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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

Joana Rocha Joana Rocha
Valerie Chazalet Valerie Chazalet
Christelle Breton Christelle Breton

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2064 浏览次数: 8377

评审: Arsalan DaudiAdam IdoineAnonymous reviewer(s)

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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) search Galactolipids (半乳糖脂) search Monogalactosyldiacylglycerol (单半乳糖基二酰基甘油) search Crystallization (结晶) search MGDG synthase (MGDG合酶) search

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

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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

植物科学 > 植物生物化学 > 蛋白质 > 分离和纯化

植物科学 > 植物生物化学 > 蛋白质 > 结构

生物化学 > 蛋白质 > 表达

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