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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

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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

  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 dehydrate (Alfa Aesar, catalog number: 36439-A3 )
  36. NiCl2 hexahydrate (Alfa Aesar, catalog number: 43185 )
  37. Polyethelene glycol 8000 (Sigma-Aldrich, catalog number: 89510 )
  38. NaSCN (VWR International, catalog number: 10118-142 )
  39. Polyethelene 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

  1. Co-transform plasmids encoding HSV-1 UL31 (15-185) or PRV UL31 (1-176), in His6-SUMO-3C-pET24b, and HSV-1 UL34 (51-306) or PRV UL34 (18-271), in pGEX-6P1, into E. coli Rosetta cells using heat shock transformation. Plate on LB-agar plates containing 100 mg/L ampicillin, 50 mg/L kanamycin and 34 mg/L chloramphenicol.
  2. Pick a single colony to inoculate a starter culture of 50 ml TB medium containing 1% glucose, 2 mM MgSO4, 34 mg/L chloramphenicol, 100 mg/L ampicillin and 100 mg/L kanamycin. Incubate the culture in a shaker at 37 °C, 200 rpm, for 16 h.
  3. Inoculate 3 L of TB containing 0.2% lactose, 2 mM MgSO4, 100 mg/L ampicillin and 100 mg/L kanamycin with 10 ml of the starter culture and grow at 37 °C, 200 rpm, for 4 h. Reduce the temperature to 25 °C and grow the cultures for another 16 h. Typically a final OD600 of 5-6 is reached.
  4. Harvest cells by centrifugation at 5,000 x g for 30 min at 4 °C.
  5. Filter and degas all buffers used in purification.
  6. Throughout the purification, confirm the presence and quality of the purified proteins by SDS-PAGE analysis and Coomassie staining (Figure 1). Determine protein concentration by measuring the OD280 [extinction coefficients: 37,360 M-1 cm-1 (HSV-1 NEC) and 34,840 M-1 cm-1 (PRV NEC)] using a spectrophotometer.
  7. Wash cell pellets in lysis buffer and centrifuge in 50 ml conical tubes for 5 min at 5,000 x g at 4 °C. Discard the supernatant and process pellets immediately or flash freeze in liquid nitrogen and store at -80 °C.
  8. Resuspend cell pellets in lysis buffer supplemented with the cOmplete Protease Inhibitor mix (one tablet/25 ml lysate) and DNase I (one tip of a spatula/50 ml lysate).
  9. Lyse cells in a microfluidizer (3 runs, 80 psi). A comparable cell disruptor may be used instead.
  10. Clear the cell lysate by centrifugation at 20,000 x g at 4 °C for 30 min. All subsequent steps are to be carried out at 4 °C or on ice.
  11. Apply the supernatant onto a Ni-NTA sepharose column (8 ml), equilibrated with lysis buffer, with a flow rate of 2 ml/min and wash with lysis buffer containing 20-40 mM imidazole. Elute bound proteins with lysis buffer containing 250 mM imidazole.
  12. Pass the eluted proteins over a GSH sepharose column (8 ml), equilibrated with lysis buffer, to remove excess His6-SUMO-UL31. After washing with lysis buffer, use PreScission protease in a 1:30 molar ratio to cleave His6-SUMO and GST tags on the GSH column for 16 h in one column volume lysis buffer circulating at a flow rate of 0.3 ml/min.
  13. Apply lysis buffer to the GSH sepharose column and collect the flow-through fraction containing untagged NEC and His6-SUMO. Apply this sample onto 2 tandem-coupled 1 ml Talon HiTrap columns to separate His6-SUMO from the NEC. Collect the flow-through fraction, containing the untagged NEC, and concentrate it using a concentrator with a MWCO of 50 kDa.
  14. Between different protein preparations, wash the Ni-NTA and GSH columns with one column volume 6 M guanidine hydrochloride and 0.2 M acetic acid. Then wash with 10 column volumes dH2O.
  15. To obtain monodisperse NEC, as a final purification step, apply the concentrated sample from step 13 to a Superdex 75 10/300 column equilibrated with gel filtration buffer.
  16. Pool NEC-containing fractions from the monodisperse peak and concentrate up to ~10 mg/ml. Aliquot protein samples and flash freeze in liquid nitrogen for subsequent storage at -80 °C. The typical yield is 10 mg monodisperse NEC per L TB culture.
  17. Prior to crystallization, dilute proteins to 5 mg/ml and filter through a 0.1 µm membrane to remove aggregates.
  18. Grow HSV-1 NEC crystals by vapor diffusion in hanging drops with 0.5 µl protein and 0.5 µl reservoir solution in 10% PEG 8000, 0.1 M Na citrate, pH 5.6, 5 mM NiCl2 at 22 °C. HSV-1 NEC crystals resembling hexagonal plates appear after 2 days and reach their final size after 1 week (Figure 2A).
  19. Soak HSV-1 NEC crystals briefly in the reservoir solution containing 25% glycerol as a cryoprotectant and flash freeze in liquid nitrogen.
  20. Grow PRV NEC crystals by vapor diffusion in hanging drops with 0.5 µl protein and 0.5 µl reservoir solution in 18% PEG 3350, 0.3 M NaSCN, 0.3 M NaCl at 22 °C. Tetragonal crystals of PRV NEC appear after one day and reach their final size after 2 days (Figure 2B).
  21. Soak PRV NEC crystals briefly in the reservoir solution containing 15% meso-erythritol as a cryoprotectant and flash freeze in liquid nitrogen.

Representative data


Figure 1. 12% SDS-PAGE analysis of pure HSV-1 and PRV NEC


Figure 2. Crystals of HSV-1 NEC (A) and PRV NEC (B). Crystals were grown in hanging drops at 22 °C. The reservoir solution for growing HVS-1 NEC crystals contained 10% PEG 8000, 0.1 M Na citrate, pH 5.6, 5 mM NiCl2, and for PRV NEC crystals 18% PEG 3350, 0.3 M NaSCN, 0.3 M NaCl.

Notes

Crystallization of HSV-1 NEC depends on the formation of a disulfide bond involving Cys278UL31. Therefore, not more than 0.5 mM TCEP may be used during purification. Both protein complexes should be purified and frozen within 40 h to prevent degradation and aggregation.

Recipes

  1. TB medium (per liter), autoclaved or filter-sterilized
    12 g tryptone
    24 g yeast extract
    4 ml glycerol
    0.231 g KH2PO4
    1.254 g K2HPO4
  2. Lysis buffer
    50 mM HEPES, pH 7.0
    500 mM NaCl
    10% glycerol
    0.5 mM TCEP
  3. Gel filtration buffer
    20 mM HEPES, pH 7.0
    100 mM NaCl
    0.5 mM TCEP

Acknowledgements

This work was funded by the NIH grants 1R21AI097573 and 1R01GM111795 (E.E.H.), the Burroughs Wellcome Fund (E.E.H.), and by the postdoctoral fellowship from the Deutsche Forschungsgemeinschaft GZ: BI 1658/1-1 (J.M.B.). Parts of this protocol were published previously in Bigalke et al., 2014 and Bigalke & Heldwein, 2015.

References

  1. Bigalke, J. M., Heuser, T., Nicastro, D. and Heldwein, E. E. (2014). Membrane deformation and scission by the HSV-1 nuclear egress complex. Nat Commun 5: 4131.
  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. Pitts, J. D., Klabis, J., Richards, A. L., Smith, G. A. and Heldwein, E. E. (2014). Crystal structure of the herpesvirus inner tegument protein UL37 supports its essential role in control of viral trafficking. J Virol 88(10): 5462-5473.

简介

该协议描述了来自单纯疱疹病毒1和伪狂犬病病毒的核出口复合物(NEC)的可溶形式的产生和结晶。 NEC是由保守蛋白UL31和UL34组成的异源二聚体。 NEC低聚使受感染细胞中的衣壳周围的内核膜变形,从而在核出口期间介导衣壳发芽进入核周空间。 我们已经成功地开发了一个协议,从大规模制备高纯度NEC从两种不同的病毒在原核表达系统,这使我们能够结晶这些病毒蛋白复合物和确定其结构。 该程序可适于纯化和结晶其它可溶性蛋白质复合物。

关键字:核出口复杂, 大肠杆菌中的蛋白质表达, 蛋白纯化, 结晶

材料和试剂

  1. Talon HiTrap柱,1ml(GE Healthcare,目录号:28-9537-66)
  2. 24孔结晶板,VDX润滑板(Hampton Research Corporation,目录号:HR3-170)
  3. 盖玻片,22mm,硅化(Hampton Research Corporation,目录号:HR3-233)
  4. 尼龙亲水膜过滤器,0.2μm(Merck Millipore Corporation,目录号:GNWP04700)
  5. Ultrafree离心过滤器,0.1μm(Merck Millipore Corporation,目录号:UFC30VV00)
  6. LB(Luria Bertani)琼脂平板上
  7. 大肠杆菌(大肠杆菌)BL21 Rosetta(DE3)(Merck Millipore Corporation,Novagen,目录号:70954)
  8. pGEX-6P1载体(GE Healthcare,目录号:28-9546-48)
  9. pET-24b载体(Merck Millipore Corporation,目录号:69750-3),修饰成包括编码His 6 -SUMO标签的序列,随后是PreScission切割位点
  10. 氨苄青霉素(Fisher Scientific,目录号:BP1760-25)
  11. 卡那霉素(Fisher Scientific,Durable,目录号:BP906-5)
  12. 氯霉素(Fisher Scientific,Bioreagents,目录号:BP904-100)
  13. 胰蛋白胨(Fisher Scientific,目录号:BP1421-2)
  14. 酵母提取物(Fisher Scientific,目录号:BP1422-2)
  15. 甘油(P212121,目录号:RP-G22020-0.5)

  16. (Fisher Scientific,目录号:P386-500)< br />

  17. (Fisher Scientific,目录号:LC200901)
  18. 葡萄糖(American Bio,目录号:AB00715)
  19. 乳糖(Fisher Scientific,Durable,目录号:L5-500)
  20. MgSO 4·7H 2 O(Sigma-Aldrich,Fluka,目录号:63140)
  21. 无EDTA的蛋白酶抑制剂混合物片剂(Sigma-Aldrich,cOmplete TM ,目录号:5056489001)
  22. 来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25-1g)
  23. HEPES(Fisher Scientific,Durable,目录号:BP310500)
  24. 氯化钠(NaCl)(Thermo Fisher Scientific,Durable,目录号:S271-10)
  25. TCEP HCl(P212121,目录号:SV-TCEP-25g)
  26. 咪唑(Thermo Fisher Scientific,ACROS Organics,目录号:301870010)
  27. Ni Sepharose 6 Fast Flow(GE Healthcare,目录号:17-5318-02)
  28. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17-0756-05)
  29. PreScission蛋白酶(内部生产;更多信息参见参考文献3)
  30. 12%Mini Protean TGX预制凝胶(Bio-Rad Laboratories,目录号:456-1046)
  31. Coomassie Brilliant Blue R-250(Bio-Rad Laboratories,目录号:161-0400)
  32. 乙酸(Fisher Scientific,Durable,目录号:A491-212)
  33. 乙醇,190标准(Fisher Scientific,Durable,目录号:04-355-226)
  34. 盐酸胍(Sigma-Aldrich,目录号:50950)
  35. 柠檬酸三钠脱水物(Alfa Aesar,目录号:36439-A3)
  36. NiCl 2六水合物(Alfa Aesar,目录号:43185)
  37. 聚乙二醇8000(Sigma-Aldrich,目录号:89510)
  38. NaSCN(VWR International,目录号:10118-142)
  39. 聚乙二醇3350(Sigma-Aldrich,目录号:P3640)
    注意:产品P3640已停产。
  40. 中赤藓糖醇(VWR International,目录号:CAAAA15813-22)
  41. TB(极好的肉汤)培养基(见配方)
  42. 裂解缓冲液(见配方)
  43. 凝胶过滤缓冲液(见配方)

设备

  1. 标准实验室设备
  2. 分光光度计装置(Thermo Fisher Scientific,NanoDrop ,型号:1000)或类似的UV/VIS分光光度计
    注意:此产品已由制造商停产。
  3. 微流化器细胞破碎仪(Microfluidics,型号:110S)或类似的细胞裂解设备
    注意:此产品已由制造商停产。
  4. 泵(Bio-Rad Laboratories,型号:EP-1) 注意:此产品已由制造商停产。
  5. 色谱系统(Bio-Rad Laboratories,DuoFlow TM ,型号:中压)或类似的色谱系统
  6. Superdex 75 10/300(GE Healthcare,目录号:17-5174-01)
  7. 立体显微镜(ZEISS,型号:SteREO Discovery V8)或相当的显微镜
  8. 带有Ultracel-50膜的Amicon Ultra-15离心过滤器单元(Merck Millipore Corporation,目录号:UFC905096)
  9. 超声波浴(Fisher Scientific,型号:FS60)或类似的超声处理装置
    注意:此产品已由制造商停用。

程序

  1. 在His6-SUMO-3C-pET24b和HSV-1UL34(51-306)或PRV UL34(18-271)中编码HSV-1UL31(15-185)或PRV UL31(1-176)的共转化质粒, ,在pGEX-6P1中,转化为E。使用热休克转化的大肠杆菌 Rosetta细胞。在含有100mg/L氨苄青霉素,50mg/L卡那霉素和34mg/L氯霉素的LB-琼脂平板上平板。
  2. 挑取单个菌落以接种含有1%葡萄糖,2mM MgSO 4,34mg/L氯霉素,100mg/L氨苄青霉素和100mg/L卡那霉素的50ml TB培养基的发酵剂培养物。在37°C,200 rpm下,在振荡器中孵育培养物16小时
  3. 用10ml起始培养物接种含有0.2%乳糖,2mM MgSO 4,100mg/L氨苄青霉素和100mg/L卡那霉素的3L TB,并在37℃,200rpm, 4小时。将温度降低至25℃,并再培养16小时。通常达到5-6的最终OD <600> 。
  4. 通过在4℃下以5,000xg离心30分钟收获细胞
  5. 过滤和脱气净化中使用的所有缓冲液。
  6. 在整个纯化过程中,通过SDS-PAGE分析和考马斯染色确认纯化蛋白质的存在和质量(图1)。通过测量OD细胞的280消光系数:37,360μMsup-1(HSV-1 NEC)和34,840μM荧光素酶来确定蛋白质浓度,使用分光光度计,测量p -1 cm -1 (PRV NEC)。
  7. 在裂解缓冲液中洗涤细胞沉淀,并在5000ml锥形管中在5,000xg在4℃下离心5分钟。弃去上清液,立即处理颗粒或在液氮中快速冷冻,并储存在-80℃
  8. 将细胞沉淀悬浮在补充有完全蛋白酶抑制剂混合物(一片/25ml裂解物)和DNase I(一个铲尖/50ml裂解物)的裂解缓冲液中。
  9. 在微流化器(3次运行,80psi)中裂解细胞。可以使用类似的细胞破碎剂。
  10. 通过在4℃下以20,000×g离心30分钟来清除细胞裂解物。所有后续步骤均应在4°C或冰上进行
  11. 将上清液施加到Ni-NTA琼脂糖柱(8ml)上,用裂解缓冲液平衡,流速为2ml/min,用含有20-40mM咪唑的裂解缓冲液洗涤。用含有250mM咪唑的裂解缓冲液洗脱结合的蛋白质。
  12. 使洗脱的蛋白通过GSH琼脂糖柱(8ml),用裂解缓冲液平衡,以除去过量的His 6 -SUMO-UL31。在用裂解缓冲液洗涤后,使用1:30摩尔比的PreScission蛋白酶,在一个柱体积裂解缓冲液中,在流速下循环,在GSH柱上切割His 6 -SUMO和GST标签16小时为0.3ml/min
  13. 将裂解缓冲液应用于GSH琼脂糖柱,并收集含有未标记的NEC和His 6 -SUMO的流通级分。将该样品应用于2个串联偶联的1ml Talon HiTrap柱,以从NEC中分离His 6 6 -SUMO。收集含有未标记NEC的流通级分,并使用MWCO为50kDa的浓缩器浓缩。
  14. 在不同蛋白质制备之间,用一柱体积6M盐酸胍和0.2M乙酸洗涤Ni-NTA和GSH柱。然后用10个柱体积的dH 2 O洗涤
  15. 为了获得单分散NEC,作为最终纯化步骤,将来自步骤13的浓缩样品应用于用凝胶过滤缓冲液平衡的Superdex 75 10/300柱。
  16. 从单分散峰收集含NEC的级分,并浓缩至?10mg/ml。等分蛋白样品并在液氮中快速冷冻,用于随后在-80℃下储存。典型产率为每L TB培养物10mg单分散NEC
  17. 在结晶之前,将蛋白质稀释至5mg/ml,并通过0.1μm膜过滤以除去聚集体
  18. 通过在悬滴中通过在10%PEG 8000,0.1M柠檬酸钠,pH 5.6,5mM NiCl 2中在22℃下的0.5μl蛋白质和0.5μl储存溶液中的蒸汽扩散来生长HSV-1 NEC晶体。 HSV-1 NEC晶体类似于六角形板,在2天后出现,并在1周后达到其最终尺寸(图2A)。
  19. 在含有25%甘油作为冷冻保护剂的储存溶液中短暂地浸泡HSV-1 NEC晶体,并在液氮中快速冷冻。
  20. 生长PRV NEC晶体通过蒸汽扩散悬滴在0.5%蛋白质和0.5升储存溶液在18%PEG 3350,0.3 M NaSCN,0.3 M NaCl在22℃。一天后出现PRV NEC的四方晶体,2天后达到最终尺寸(图2B)。
  21. 在含有15%内消旋赤藓醇作为冷冻保护剂的储液中短暂浸泡PRV NEC晶体,并在液氮中快速冷冻。

代表数据


图1.纯HSV-1和PRV NEC 的12%SDS-PAGE分析


图2. HSV-1 NEC(A)和PRV NEC(B)的晶体。晶体在22℃下悬滴生长。用于生长HVS-1 NEC晶体的储存溶液含有10%PEG 8000,0.1M柠檬酸钠,pH 5.6,5mM NiCl 2以及用于PRV NEC晶体的18%PEG 3350,0.3M NaSCN, 0.3 M NaCl。

笔记

HSV-1 NEC的结晶取决于涉及Cys278 sub-UL31的二硫键的形成。因此,在纯化过程中可以使用不超过0.5mM的TCEP。两种蛋白质复合物应该在40小时内被纯化和冷冻以防止降解和聚集。

食谱

  1. TB培养基(每升),高压灭菌或过滤灭菌
    12克胰蛋白胨
    24g酵母提取物
    4ml甘油 0.231g KH 2 PO 4 sub/
    1.254g K 2 HPO 4
  2. 裂解缓冲液
    50mM HEPES,pH7.0 500 mM NaCl
    10%甘油 0.5 mM TCEP
  3. 凝胶过滤缓冲液
    20mM HEPES,pH7.0 100 mM NaCl
    0.5 mM TCEP

致谢

这项工作由NIH赠款1R21AI097573和1R01GM111795(E.E.H.),Burroughs Wellcome基金(E.E.H)和来自Deutsche Forschungsgemeinschaft GZ:BI 1658/1-1(J.M.B.)的博士后研究金资助。该方案的一部分先前在Bigalke等人,2014和Bigalke& Heldwein,2015。

参考文献

  1. Bigalke,JM,Heuser,T.,Nicastro,D。和Heldwein,EE(2014)。  由HSV-1核出口复合物引起的膜变形和分裂。 Nat Commun 5:4131.
  2. Bigalke,JM和Heldwein,EE(2015)。  结构通过疱疹病毒的核出口复合物的膜出芽的基础.EMEM EMBO杂志34(23):2921-2936。
  3. Pitts,JD,Klabis,J.,Richards,AL,Smith,GA and Heldwein,EE(2014)。  疱疹病毒内膜蛋白UL37的晶体结构支持其在控制病毒性运输中的重要作用。 :5462-5473。
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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.
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