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

Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)

疱疹病毒细胞核出口复合体（NEC）的表达、纯化和结晶

Janna M. Bigalke Janna M. Bigalke
Ekaterina E. Heldwein Ekaterina E. Heldwein

发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1872 浏览次数: 9050

评审: Arsalan DaudiBalasubramanian VenkatakrishnanAnonymous 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 The EMBO Journal, featuring study using the protocol.

Dec 2015

Advertisement

Technologies for RNA Detection
High-throughput T-cell antigen discovery technology platform and its applications
Special Issue on Protocols for Organoid Research
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

利用MViewEMA高效评估蛋白质复合物结构模型整体准确性的分步操作方法

利用MViewEMA高效评估蛋白质复合物结构模型整体准确性的分步操作方法

Lei Xie [...] Guijun Zhang

2026年08月20日 431 阅读

肌动蛋白丝末端结构的冷冻电镜分析流程

肌动蛋白丝末端结构的冷冻电镜分析流程

Nicholas J. Palmer and Roberto Dominguez

2026年09月05日 594 阅读

用于高盐极端微生物核糖体分析的微球菌核酸酶纯化方法

用于高盐极端微生物核糖体分析的微球菌核酸酶纯化方法

Pavlina Gregorova [...] Peter L. Sarin

2026年09月20日 192 阅读

Abstract

The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.

Keywords: Nuclear egress complex (核出口复杂) search Protein expression in E. coli (大肠杆菌中的蛋白质表达) search Protein purification (蛋白纯化) search Crystallization (结晶) search

Materials and Reagents

  1. Talon HiTrap columns, 1 ml (GE Healthcare, catalog number: 28-9537-66 )
  2. 24-well crystallization plates, VDX Greased Plates (Hampton Research Corporation, catalog number: HR3-170 )
  3. Cover slips, 22 mm, siliconized (Hampton Research Corporation, catalog number: HR3-233 )
  4. Nylon hydrophilic membrane filters, 0.2 µm (Merck Millipore Corporation, catalog number: GNWP04700 )
  5. Ultrafree centrifugal filters, 0.1 µm (Merck Millipore Corporation, catalog number: UFC30VV00 )
  6. LB (Luria Bertani) agar plates
  7. Escherichia coli (E. coli) BL21 Rosetta (DE3) (Merck Millipore Corporation, Novagen, catalog number: 70954 )
  8. pGEX-6P1 vector (GE Healthcare, catalog number: 28-9546-48 )
  9. pET-24b vector (Merck Millipore Corporation, catalog number: 69750-3 ), modified to include a sequence encoding a His6-SUMO tag followed by a PreScission cleavage site
  10. Ampicillin (Fisher Scientific, catalog number: BP1760-25 )
  11. Kanamycin (Fisher Scientific, Durable, catalog number: BP906-5 )
  12. Chloramphenicol (Fisher Scientific, Bioreagents, catalog number: BP904-100 )
  13. Tryptone (Fisher Scientific, catalog number: BP1421-2 )
  14. Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
  15. Glycerol (P212121, catalog number: RP-G22020-0.5 )
  16. KH2PO4 (Fisher Scientific, catalog number: P386-500 )
  17. K2HPO4 (Fisher Scientific, catalog number: LC200901 )
  18. Glucose (American Bio, catalog number: AB00715 )
  19. Lactose (Fisher Scientific, Durable, catalog number: L5-500 )
  20. MgSO4·7H2O (Sigma-Aldrich, Fluka, catalog number: 63140 )
  21. EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich, cOmpleteTM, catalog number: 5056489001 )
  22. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25-1 g )
  23. HEPES (Fisher Scientific, Durable, catalog number: BP310500 )
  24. Sodium chloride (NaCl) (Thermo Fisher Scientific, Durable, catalog number: S271-10 )
  25. TCEP HCl (P212121, catalog number: SV-TCEP-25 g )
  26. Imidazole (Thermo Fisher Scientific, ACROS Organics, catalog number: 301870010 )
  27. Ni Sepharose 6 Fast Flow (GE Healthcare, catalog number: 17-5318-02 )
  28. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17-0756-05 )
  29. PreScission protease (produced in house; for more information see Reference 3)
  30. 12% Mini Protean TGX precast gels (Bio-Rad Laboratories, catalog number: 456-1046 )
  31. Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, catalog number: 161-0400 )
  32. Acetic acid (Fisher Scientific, Durable, catalog number: A491-212 )
  33. Ethanol, 190 proof (Fisher Scientific, Durable, catalog number: 04-355-226 )
  34. Guanidine hydrochloride (Sigma-Aldrich, catalog number: 50950 )
  35. Trisodium citrate dihydrate (Alfa Aesar, catalog number: 36439-A3 )
  36. NiCl2 hexahydrate (Alfa Aesar, catalog number: 43185 )
  37. Polyethylene glycol 8000 (Sigma-Aldrich, catalog number: 89510 )
  38. NaSCN (VWR International, catalog number: 10118-142 )
  39. Polyethylene glycol 3350 (Sigma-Aldrich, catalog number: P3640 )
    Note: Product P3640 has been discontinued.
  40. Meso-erythritol (VWR International, catalog number: CAAAA15813-22 )
  41. TB (Terrific broth) medium (see Recipes)
  42. Lysis buffer (see Recipes)
  43. Gel filtration buffer (see Recipes)

Equipment

  1. Standard laboratory equipment
  2. Spectrophotometer device (Thermo Fisher Scientific, NanoDropTM, model: 1000 ) or comparable UV/VIS spectrophotometer
    Note: This product has been discontinued by the manufacturer.
  3. Microfluidizer cell disruptor (Microfluidics, model: 110S ) or comparable cell lysis equipment
    Note: This product has been discontinued by the manufacturer.
  4. Pumps (Bio-Rad Laboratories, model: EP-1 )
    Note: This product has been discontinued by the manufacturer.
  5. Chromatography system (Bio-Rad Laboratories, DuoFlowTM, model: Medium-pressure ) or comparable chromatography system
  6. Superdex 75 10/300 (GE Healthcare, catalog number: 17-5174-01 )
  7. Stereo microscope (ZEISS, model: SteREO Discovery V8 ) or comparable microscope
  8. Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane (Merck Millipore Corporation, catalog number: UFC905096 )
  9. Ultrasonic bath (Fisher Scientific, model: FS60) or comparable sonication device
    Note: This product has been discontinued by the manufacturer. 

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

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Bigalke, J. M. and Heldwein, E. E. (2016). Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC) . Bio-protocol 6(14): e1872. DOI: 10.21769/BioProtoc.1872.
  2. Bigalke, J. M. and Heldwein, E. E. (2015). Structural basis of membrane budding by the nuclear egress complex of herpesviruses. EMBO J 34(23): 2921-2936.

    3. ris ris Download Citation in RIS Format

分类

生物化学 > 蛋白质 > 分离和纯化

生物化学 > 蛋白质 > 表达

生物化学 > 蛋白质 > 结构

您对这篇实验方案有问题吗？

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