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
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1872 浏览次数: 8977
评审: Arsalan DaudiBalasubramanian VenkatakrishnanAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
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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)
Protein expression in E. coli (大肠杆菌中的蛋白质表达)
Protein purification (蛋白纯化)
Crystallization (结晶)
Materials and Reagents
- Talon HiTrap columns, 1 ml (GE Healthcare, catalog number: 28-9537-66 )
- 24-well crystallization plates, VDX Greased Plates (Hampton Research Corporation, catalog number: HR3-170 )
- Cover slips, 22 mm, siliconized (Hampton Research Corporation, catalog number: HR3-233 )
- Nylon hydrophilic membrane filters, 0.2 µm (Merck Millipore Corporation, catalog number: GNWP04700 )
- Ultrafree centrifugal filters, 0.1 µm (Merck Millipore Corporation, catalog number: UFC30VV00 )
- LB (Luria Bertani) agar plates
- Escherichia coli (E. coli) BL21 Rosetta (DE3) (Merck Millipore Corporation, Novagen, catalog number: 70954 )
- pGEX-6P1 vector (GE Healthcare, catalog number: 28-9546-48 )
- 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
- Ampicillin (Fisher Scientific, catalog number: BP1760-25 )
- Kanamycin (Fisher Scientific, Durable, catalog number: BP906-5 )
- Chloramphenicol (Fisher Scientific, Bioreagents, catalog number: BP904-100 )
- Tryptone (Fisher Scientific, catalog number: BP1421-2 )
- Yeast extract (Fisher Scientific, catalog number: BP1422-2 )
- Glycerol (P212121, catalog number: RP-G22020-0.5 )
- KH2PO4 (Fisher Scientific, catalog number: P386-500 )
- K2HPO4 (Fisher Scientific, catalog number: LC200901 )
- Glucose (American Bio, catalog number: AB00715 )
- Lactose (Fisher Scientific, Durable, catalog number: L5-500 )
- MgSO4·7H2O (Sigma-Aldrich, Fluka, catalog number: 63140 )
- EDTA-free protease inhibitor cocktail tablets (Sigma-Aldrich, cOmpleteTM, catalog number: 5056489001 )
- Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25-1 g )
- HEPES (Fisher Scientific, Durable, catalog number: BP310500 )
- Sodium chloride (NaCl) (Thermo Fisher Scientific, Durable, catalog number: S271-10 )
- TCEP HCl (P212121, catalog number: SV-TCEP-25 g )
- Imidazole (Thermo Fisher Scientific, ACROS Organics, catalog number: 301870010 )
- Ni Sepharose 6 Fast Flow (GE Healthcare, catalog number: 17-5318-02 )
- Glutathione Sepharose 4B (GE Healthcare, catalog number: 17-0756-05 )
- PreScission protease (produced in house; for more information see Reference 3)
- 12% Mini Protean TGX precast gels (Bio-Rad Laboratories, catalog number: 456-1046 )
- Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, catalog number: 161-0400 )
- Acetic acid (Fisher Scientific, Durable, catalog number: A491-212 )
- Ethanol, 190 proof (Fisher Scientific, Durable, catalog number: 04-355-226 )
- Guanidine hydrochloride (Sigma-Aldrich, catalog number: 50950 )
- Trisodium citrate dihydrate (Alfa Aesar, catalog number: 36439-A3 )
- NiCl2 hexahydrate (Alfa Aesar, catalog number: 43185 )
- Polyethylene glycol 8000 (Sigma-Aldrich, catalog number: 89510 )
- NaSCN (VWR International, catalog number: 10118-142 )
- Polyethylene glycol 3350 (Sigma-Aldrich, catalog number: P3640 )
Note: Product P3640 has been discontinued. - Meso-erythritol (VWR International, catalog number: CAAAA15813-22 )
- TB (Terrific broth) medium (see Recipes)
- Lysis buffer (see Recipes)
- Gel filtration buffer (see Recipes)
Equipment
- Standard laboratory equipment
- Spectrophotometer device (Thermo Fisher Scientific, NanoDropTM, model: 1000 ) or comparable UV/VIS spectrophotometer
Note: This product has been discontinued by the manufacturer. - Microfluidizer cell disruptor (Microfluidics, model: 110S ) or comparable cell lysis equipment
Note: This product has been discontinued by the manufacturer. - Pumps (Bio-Rad Laboratories, model: EP-1 )
Note: This product has been discontinued by the manufacturer. - Chromatography system (Bio-Rad Laboratories, DuoFlowTM, model: Medium-pressure ) or comparable chromatography system
- Superdex 75 10/300 (GE Healthcare, catalog number: 17-5174-01 )
- Stereo microscope (ZEISS, model: SteREO Discovery V8 ) or comparable microscope
- Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-50 membrane (Merck Millipore Corporation, catalog number: UFC905096 )
- Ultrasonic bath (Fisher Scientific, model: FS60) or comparable sonication device
Note: This product has been discontinued by the manufacturer.
Procedure
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:
- 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.
- 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.
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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
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