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Protein Expression, Purification and Crystallization of the Sxl-Unr-msl2 Ribonucleoprotein Complex

Sxl-Unr-msl2 核糖核蛋白复合体的蛋白质表达、纯化和结晶

Janosch Hennig Janosch Hennig
MS Michael Sattler

发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1917 浏览次数: 9470

评审: Arsalan DaudiLongping Victor TseAnonymous reviewer(s)

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Abstract

This protocol describes the expression, purification and crystallization of a ternary protein-protein-RNA complex, consisting of the two RNA recognition motifs (RRMs) of Sex-lethal (Sxl), the first of five cold shock domains of Upstream-of-N-Ras (Unr), and an 18-nucleotide region of msl2 mRNA, called the F fragment (Hennig et al., 2014).The biological role of the complex is the translational repression of msl2 mRNA, preventing the formation of the dosage compensation complex and subsequent 2-fold hypertranscription of X-linked genes in Drosophila females. As orthologous RRM-containing proteins and Unr exist in humans, similar complexes potentially also form during translational repression in vertebrates. The protocol describes the in vitro assembly of the complex and its purification followed by crystallization for X-ray crystallography structure determination. Part of the protocol has been published elsewhere (Hennig et al., 2013 and 2014), but some parts are described here in more detail.

Materials and Reagents

  1. Membrane dialysis tubing Spectra/Por 6 prewetted, 1.1 ml/cm 3,500 Da cut-off (Spectrum, Spectra/Por®, catalog number: 132590 )
  2. Membrane dialysis tubing Spectra/Por 6 prewetted, 1.1 ml/cm 10,000 Da cut-off (Spectrum, Spectra/Por®, catalog number: 128118 )
  3. Amicon® Ultra-15 centrifugal filter unit with Ultracel-3 membrane (Merck Millpore, catalog number: UFC900308 )
  4. Amicon® Ultra-15 centrifugal filter unit with Ultracel-10 membrane (Merck Millpore, catalog number: UFC901008 )
  5. Amicon® Ultra-4 centrifugal filter unit with Ultracel-3 membrane (Merck Millpore, catalog number: UFC800324 )
  6. Amicon® Ultra-4 centrifugal filter unit with Ultracel-10 membrane (Merck Millpore, catalog number: UFC801024 )
  7. EasyXtal 15-well tool (Qiagen, catalog number: 132006 )
  8. 0.22 µm filter
  9. 96-well plate
  10. SS34 centrifugation tubes
  11. Escherichia coli (E. coli) Bl21 (DE3)from stocks (stored at -80 °C)
  12. pTRX-Sxl and pTRX-CSD plasmids (available upon request)
  13. Tobacco etch virus (TEV) protease (1 mg/ml stock solution, in-house production)
  14. Precision plus protein all blue prestained protein standard (Bio-Rad Laboratories, catalog number: 1610373 )
  15. LB medium
  16. Kanamycin (33 mg/ml stock solution, stored at -20 °C)
  17. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) (1 M stock solution)
  18. Ni-NTA resin (Qiagen, catalog number: 30230 )
  19. Sodium phosphate (molecular biology grade)
  20. Sodium chloride (molecular biology grade)
  21. Potassium phosphate (molecular biology grade)
  22. Imidazole (molecular biology grade, > 99%)
  23. β-mercaptoethanol
  24. Sodium azide
  25. Ammonium sulfate (molecular biology grade, > 99% purity)
  26. 18-mer RNA based on E fragment of msl2 mRNA: UUU UUU UGA GCA CGU GAA (synthesized) (IBA) (UniProt Consortium, catalog number: P50534 )
  27. Lithium sulfate (X-ray crystallography grade, > 99% purity)
  28. PEG 3350 (Sigma-Aldrich, catalog number: 39713 )
  29. Dithiothreitol (DTT) (> 99% purity)
  30. Glycerol (Sigma-Aldrich, catalog number: 49767 )
  31. Lysis buffer (see Recipes)
  32. Wash buffer A (see Recipes)
  33. Wash buffer B (see Recipes)
  34. Elution buffer (see Recipes)
  35. NMR buffer (see Recipes)
  36. Running buffer (see Recipes)

Equipment

  1. Erlenmeyer flasks (500 ml)
  2. FPLC protein purifier
  3. Superdex S75 gel filtration size-exclusion chromatography column HiLoad 26/600 (GE Healthcare)
  4. Nanodrop system  

Procedure

English
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文章信息

版权信息

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

如何引用

Hennig, J. and Sattler, M. (2016). Protein Expression, Purification and Crystallization of the Sxl-Unr-msl2 Ribonucleoprotein Complex. Bio-protocol 6(17): e1917. DOI: 10.21769/BioProtoc.1917.

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分类

生物化学 > 蛋白质 > 结构

生物化学 > RNA > RNA-蛋白质相互作用

生物化学 > 蛋白质 > 分离和纯化

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