Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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: 10151

评审: Arsalan DaudiLongping Victor TseAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Nature, featuring study using the protocol.

Nov 2014

Advertisement

Lonza电转系列完整产品手册
Bio-protocol welcomes Protocols in Bioinformatics and Computational Biology
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

体外RNA免疫沉淀(vitRIP)鉴定纯化蛋白的RNA结合特异性

体外RNA免疫沉淀(vitRIP)鉴定纯化蛋白的RNA结合特异性

Marisa Müller [...] Peter B. Becker

2021年03月05日 4514 阅读

黑腹果蝇中蛋白质-蛋白质相互作用和 RNA 富集的免疫沉淀

黑腹果蝇中蛋白质-蛋白质相互作用和 RNA 富集的免疫沉淀

Giulia Romano [...] Fabian Feiguin

2021年12月05日 3223 阅读

昆虫化学感受蛋白的功能注释与结构建模分步计算流程

昆虫化学感受蛋白的功能注释与结构建模分步计算流程

Rajeswari Kalepu [...] Nor Azlan Nor Muhammad

2025年11月20日 1435 阅读

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
中文翻译

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.

ris ris Download Citation in RIS Format

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

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件