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Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
Hsp90-Cdc37-Cdk4激酶复合物在酿酒酵母中的表达和纯化   

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Abstract

Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.

Keywords: Hsp90(HSP90), Cdc37(Cdc37), Cdk4(Cdk4), Chaperone(伴侣), Kinase(激酶), 2A peptides(2A肽), Yeast protein expression(酵母蛋白表达)

Background

Robustly forming complexes between Hsp90 molecular chaperone and its client kinases has proven to be refractory in vitro. Previous work indicated that overexpression of Hsp90’s co-chaperone Cdc37 together with a client kinase in insect Sf9 cells led to a stable complex between Sf9 Hsp90, exogenous Cdc37 and exogenous kinase (Vaughan et al., 2006). However, insect cell culture requires special equipment, is more difficult to genetically manipulate and is significantly slower to both grow and clone into than other well studied expression systems, like bacteria and yeast. Co-expression of the above proteins in E. coli did not yield soluble kinase/stable complex. We reasoned that Saccharomyces cerevisiae would possess the necessary machinery to help fold and facilitate the complex formation and sought to generate the complex between human Hsp90 beta, human Cdc37 and human Cdk4 kinase by co-expressing these proteins in S. cerevisiae. To attain stoichiometric expression of the three proteins, we utilized viral 2A peptides, which allowed transcription of the three proteins on one mRNA with subsequent cleaving at the translation stage. This system has been utilized in human cell lines and in rabbit reticulolysates (Kim et al., 2011; Minskaia and Ryan, 2013), but to our knowledge this is the first utilization of 2A viral expression peptides in S. cerevisiae.

Materials and Reagents

    Generating the co-expression construct
    1. 100 x 15 mm Petri dishes (Fisher Scientific, catalog number: FB0875713 )
    2. 15 ml culture tubes (Corning, Falcon®, catalog number: 352051 )
    3. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129 )
    4. 0.2 μm filter
    5. 83nu vector (obtained from Arkin lab)
    6. GeneArt Strings DNA (Thermo Fisher Scientific)
    7. NEB Builder Assembly Tool (Online: http://nebuilder.neb.com/)
    8. Gibson Assembly Cloning Kit (New England Biolabs, catalog number: E5510S )
    9. DpnI
    10. PCR Clean up Kit (Promega, catalog number: A9281 )
    11. Miniprep Kit (Omega Bio-tek, catalog number: D6942-02 )
    12. Q5 Site-Directed Mutagenesis Kit (New England Biolabs, catalog number: E0554S )
    13. Bacto-tryptone (BD, BactoTM, catalog number: 211705 )
    14. Carbenicillin (Gold Bio, catalog number: C-103-100 )
    15. Yeast extract (BD, BactoTM, catalog number: 212750 )
    16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-10KG )
    17. Agar (BD, DifcoTM, catalog number: 281210 )
    18. LB (see Recipes)
    19. LB agar (see Recipes)

  1. Expression of the Hsp90/Cdc37/Cdk4
    1. Disposable spectrophotometer cells (Agilent Technologies, catalog number: 6610018700 )
    2. JEL1 strain of Saccharomyces cereviase (MAT-alpha, leu2 trp1 ura3-52 prb1-1122 pep4-3 deltahis3::PGAL10-GAL4). Gift from Luke Rice, UT Southwestern
    3. Frozen-EZ Yeast Transformation II Kit (ZYMO RESEARCH, catalog number: T2001 )
    4. Galactose (Sigma-Aldrich, catalog number: G0625 )
    5. Yeast nitrogen base (YNB) (BD, DifcoTM, catalog number: 291940 )
    6. Glucose (Sigma-Aldrich, catalog number: G8270 )
    7. CSM-His amino acid mixture (MP Biomedicals, catalog number: 4510-312 )
    8. Peptone (BD, BactoTM, catalog number: 211820 )
    9. Yeast extract (BD, BactoTM, catalog number: 212750 )
    10. Sodium DL-lactate solution (Sigma-Aldrich, catalog number: L1375 )
    11. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    12. SD-His (see Recipes)
    13. YPGL media (see Recipes)

  2. Purification of the Hsp90/Cdc37/Cdk4 complex
    1. 30 ml syringe (BD, catalog number: 309650 ) with 16 G needle (BD, catalog number: 305196 )
    2. 50 ml conical tubes (Corning, catalog number: 352070 )
    3. Concentrators, 15 ml, 30 kDa cutoff (EMD Millipore, catalog number: UFC903096 )
    4. Dialysis tubing, 10 kDa cutoff (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 68100 )
    5. 12 x 75 mm tubes for fraction collection (Fisher Scientific, catalog number: 14-959-16 )
    6. FLAG peptide (GenScript, catalog number: RP10586 )
    7. Protease inhibitors, EDTA free (Roche Diagnostics, catalog number: 11873580001 )
    8. Ni-NTA Superflow beads (QIAGEN, catalog number: 30450 )
    9. Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich, catalog number: M8823 )
    10. TEV protease, prepped in lab, 10 mg/ml, same as (Sigma-Aldrich, catalog number: T4455 )
    11. Liquid nitrogen
    12. Bolt 4-12% Bis-Tris Plus Gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: NW04120BOX )
    13. NuPAGE LDS sample buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0007 )
    14. NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0001 )
    15. Trizma base (Sigma-Aldrich, catalog number: T1503 )
    16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-10KG )
    17. Imidazole (Sigma-Aldrich, catalog number: I2399 )
    18. Magnesium chloride (MgCl2), 1 M stock (Sigma-Aldrich, catalog number: 63069 )
    19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    20. Sodium molybdate (NaMoO4) (Sigma-Aldrich, catalog number: 243655 )
    21. Dithiothreitol (DTT) (Gold Bio, catalog number: DTT100 )
    22. Lysis buffer (see Recipes)
    23. Dialysis buffer (see Recipes)
    24. Gel filtration buffer (see Recipes)

Equipment

  1. Generating the co-expression construct
    1. PCR machine (like Biorad DNA Engine BG96TC)
    2. 37 °C culture shaker (like New Brunswick Innova 43 series)
    3. 4 °C and -20 °C fridge (any brand)
    4. 42 °C heat block (like Thomas Scientific 3661044)

  2. Expression of Hsp90/Cdc37/Cdk4
    1. 2.5 L flasks (Ultra Yield) (Thomson Instrument, catalog number: 931136-B )
    2. Autoclave
    3. Centrifuge capable of 3,000 x g utilizing 1 L bottles (like Beckman Coulter, model: Avanti® J20 Series )
    4. 30 °C culture shaker capable of shaking at 200 rpm 250 ml and 1 L flasks (like Eppendorf, New BrunswickTM, model: Innova® 4200 series)
    5. UV-Visible spectrophotometer (Agilent Technologies, model: Agilent 8453 )

  3. Purification of the Hsp90/Cdc37/Cdk4 complex
    1. EmulsiFlex-C3 (Avestin, model: EmulsiFlex-C3 )
    2. Centrifuge (like Beckman Coulter, model: Avanti® J20 Series )
    3. High-speed centrifuge tubes (capable of 30,000 x g like Beckman Coulter, catalog number: 357002 )
    4. Rocker (like Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88880019 )
    5. MonoQ 10/100GL column (GE Healthcare, catalog number: 17-5167-01 )
    6. HiLoad 16/600 Superdex 200pg column (GE Healthcare, catalog number: 28989335 )
    7. Glass gravity columns (Bio-Rad Laboratories, catalog number: 7372512 )
    8. ÄKTApurifier system (GE Healthcare, model: ÄKTApurifier system)
    9. -80 °C fridge (like Eppendorf, New BrunswickTM, model: U570 ULT )
    10. Assortment of beaker sizes
    11. Magnetic stand (like EMD Millipore, catalog number: LSKMAGS15 )
    12. Mini Gel tank (Thermo Fisher Scientific, catalog number: A25977 )
    13. PowerEase 500 Power Supply (Thermo Fisher Scientific, model: PowerEase® 500 )
    14. Liquid nitrogen flask (like Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2129 )
    15. Spatulas (Cole-Parmer, catalog number: UX-06369-16 )

Procedure

  1. Generating the co-expression construct
    1. To co-express human Hsp90β, human Cdc37 and human Cdk4 we utilized viral 2A peptides. This way we were able to construct a single plasmid, which had all three proteins in it. (see Figure 1) The exact 2A sequence we used (referred to as P2A) was sourced from Porcine Teschovirus-1 and was GSGATNFSLLKQAGDVEENPGP. The resulting construct was of this arrangement: hCdc37-TEVsite-P2A-hHsp90β-TEVsite-FLAG-P2A-hCdk4-TEVsite-HisTag. The nucleotide sequence which was inserted between hCdc37 and hHsp90 was: CAGAACCTGTACTTTCAGGGCGGATCCGGTGCCACCAACTTTAGCTTGTTGAAGCAGGCTGGAGACGTGGAGGAAAATCCTGGACCC, between hHsp90 and hCdk4 was: CAAAACTTATACTTCCAGGGTGACTATAAGGACGATGACGACAAAGGCTCCGGTGCCACCAACTTCTCATTATTGAAACAGGCCGGTGATGTAGAGGAAAATCCAGGACCT, and after hCdk4 was: CAGAATCTGTATTTTCAGGGACATCATCACCATCACCATTAATGA. The complete sequence above was codon optimized (the regions between proteins were manually checked to be dissimilar to ease cloning). See Appendix for complete insert sequence.


      Figure 1. Design and cloning of the construct with Hsp90, Cdc37 and Cdk4 in it

    2. The sequence was then split into two pieces (at nucleotide number 2,212) and both parts were ordered as GeneArt Strings DNA from Thermo Fisher Scientific. Using NEBuilder, primers were designed to amplify the 83nu vector and each of the GeneArt Strings DNA as to clone them using Gibson Assembly Cloning Kit.
    3. 83nu vector and each of the GeneArt Strings DNA are amplified using a PCR machine, then digested with DpnI at 37 °C overnight (O/N) (~16 h), PCR cleaned up, assembled using Gibson Assembly Cloning Kit and transformed into E. coli TOP10 cells supplied with the kit, which are plated and grown O/N at 37 °C on LB-carbenicillin plates.
    4. Next day, pick 10 colonies and start a 5 ml culture of LB together with 100 mg/L of carbenicillin with each colony.
    5. After 24 h growth at 37 °C with shaking, the plasmid DNA is extracted utilizing miniprep kit and sent for sequencing (tiling every 500 bp through the whole gene). In our case, the best clone had one mutation due to synthesis errors, which was corrected utilizing Q5 Site Directed Mutagenesis Kit and the protocol therein.
    6. The final construct is sequenced and stored in distilled nuclease free water at -20 °C for future use. 4 °C fridge is used for short-term storage of DNA (under a week).

  2. Expression of the Hsp90/Cdc37/Cdk4
    1. The plasmid is transformed into JEL1 (MAT-alpha, leu2 trp1 ura3-52 nprb1-1122 pep4-3 deltahis3::PGAL10-GAL4) yeast strain using Frozen-EZ Yeast Transformation II Kit protocol and plated on SD-His plates.
    2. After 3 days, pick a colony and inculcate into 100 ml SD-His media in a 250 ml flask and grow at 30 °C O/N shaking at 200 rpm.
    3. Next day, inoculate 6 x 1 L flasks of YPGL media (autoclaved) each with 10 ml of the O/N culture.
    4. After about 24 h at 30 °C and 200 rpm (optical density [OD] of 1, measured on the spectrophotometer), add powder galactose to a final concentration of 2% w/v to induce protein expression.
    5. The culture is pelleted after 6 h of growth at 3,000 x g for 20 min.
    6. Pellets are scooped up and frozen at -80 °C.

  3. Purification of the Hsp90/Cdc37/Cdk4 complex
    1. Resuspend the cell pellets in lysis buffer with protease inhibitors first with spatula, then pass the sample through a 16 G syringe needle (about 160 ml total for 6 L yeast culture).
    2. Then pass the lysate through EmulsiFlex-C3 machine at 25,000 psi, 5 complete passes for lysis following standard manufacturer’s procedures.
    3. Clear the lysate by centrifuging at 30,000 x g for 30 min.
    4. Equilibrate 5 ml bed volume of Ni-NTA beads with 20 bed volumes (100 ml) of lysis buffer at room temperature utilizing a glass gravity column. The supernatant from the centrifuge spin is added to the beads, and then rocked for 1 h at 4 °C in the capped glass gravity column.
    5. After 1 h incubation, wash the beads with 20 bed volumes of lysis buffer and then elute into 10 bed volumes of lysis buffer + 500 mM imidazole, utilizing a glass gravity column for both steps.
    6. Then incubate the resulting eluate with pre-equilibrated (10 bed volumes of lysis buffer utilizing the magnetic stand) Anti-FLAG M2 Magnetic Beads (1 ml bed volume) in a 50 ml plastic conical tube for 1 h at 4 °C, rocking.
    7. Wash the beads with 10 bed volumes of lysis buffer utilizing the magnetic stand and a 15 ml tube, and elute the sample with 3 bed volumes of lysis buffer with 75 μg/ml of FLAG peptide, twice, 25 min each time
    8. Add TEV protease to the eluent (2.5 mg) and dialyze sample against 4 L of dialysis buffer O/N utilizing dialysis tubing.
    9. Dilute the sample 1:1 with the dialysis buffer without NaCl and load onto pre-equilibrated MonoQ 10/100GL column on an ÄKTApurifier system.
    10. After washing out the unbound sample (about 3 CV [column volumes]), a gradient is run up to 1 M NaCl (over 20 CV) with fractionation to elute the bound complex (comes off at about 25% conductivity).
    11. The fractions are pooled, concentrated and then loaded on HiLoad 16/600 Superdex 200pg column pre-equilibrated in gel filtration buffer.
    12. The peak fractions (at about 0.5 CV) are pooled, concentrated, flash frozen in liquid nitrogen and stored at -80 °C. At various points (see Figure 2) during the purification aliquots are taken to be run on SDS-PAGE gel (Figure 2).


      Figure 2. SDS-PAGE gels of Hsp90-Cdc37-Cdk4 purification from yeast. A. SDS-PAGE gels show aliquots at different steps during the purification. Cell lysate–right after step C2 of purification, Supernatant–supernatant at step C3, His beads flow-through–flow-through at step C5, His beads elution–elution at step C5, Flag beads flow through–flow through of flag beads at step C6, Flag beads elution–elution at step 7. B. Two gels are fractions off MonoQ 10/100GL column and size exclusion column runs.

Data analysis

Sample prepared in this way was subsequently prepared for cryo electron microscopy (cryoEM) followed by data collection and reconstruction. The protocols utilized for these steps are described in great detail in Verba et al., 2016.

Notes

  1. Scaling up the purification Genscript Anti-DYKDDDDK G1 affinity resin (L00432) was also used successfully with glass gravity column instead of Anti-FLAG M2 Magentic Beads. Use the same amount of beads/proportion of wash/elution buffers.
  2. This protocol has also been successfully used for expression and purification of Hsp90/Cdc37/Her2 complex.
  3. Expression of kinase alone, without Hsp90/Cdc37 yields soluble, active kinase.

Recipes

  1. LB
    10 g Bacto-tryptone
    5 g yeast extract
    10 g NaCl
    in 1 L of MilliQ water
    Autoclave
    If adding antibiotic, like carbenicillin, add 50 mg/ml stock solution (0.2 μm sterile filtered) to the LB cooled to below 50 °C, to a final concentration of 50 mg/L
  2. LB agar
    As above with 15 g agar
    If adding antibiotic, like carbenicillin, add 50 mg/ml stock solution (0.2 μm sterile filtered) to the LB cooled to below 50 °C, to a final concentration of 50 mg/L
  3. SD-His
    6.7 g of yeast nitrogen base
    5 g glucose
    0.77 g CSM-His amino acid mix
    in 1 L of MilliQ water (or 100 ml for 10x)
    Sterile filter
    For plates proceed as above, but with 15 g of agar per liter
  4. YPGL media
    2% peptone
    1% yeast extract
    3% glycerol
    2% lactate
    in 1 L of deionized water
    Autoclave
  5. Lysis buffer
    20 mM Tris pH 7.5
    150 mM NaCl
    20 mM imidazole
    10 mM MgCl2
    10 mM KCl
    20 mM NaMoO4
  6. Dialysis buffer
    20 mM Tris pH 7.5
    100 mM NaCl
    10 mM MgCl2
    10 mM KCl
    20 mM NaMoO4
  7. Gel filtration buffer
    20 mM Tris pH 7.5
    150 mM NaCl
    10 mM KCl
    20 mM NaMoO4
    1 mM DTT

Acknowledgments

We thank Akihiko Arakawa (Yokoyama Lab) for sharing their insect cell purification protocol, Yao Fan (Arkin Lab) for the yeast expression vector, and D.A.A. lab members for helpful discussions, specifically Miguel Betegon for the suggestion of using viral 2A peptides and Michelle Moritz for yeast expression strain. Support for this work was provided by PSI-Biology grant U01 GM098254 (to D.A.A.), AACR-BCRF Grant 218084 for Translational Breast Cancer Research (to D.A.A.), The Cabala Family gift (to D.A.A.), HHMI International Student Research Fellowship (to K.V.) and the Howard Hughes Medical Institute (to D.A.A.).

References

  1. Kim, J. H., Lee, S. R., Li, L. H., Park, H. J., Park, J. H., Lee, K. Y., Kim, M. K., Shin, B. A. and Choi, S. Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6(4): e18556.
  2. Minskaia, E. and Ryan, M. D. (2013). Protein coexpression using FMDV 2A: effect of "linker" residues. Biomed Res Int 2013: 291730.
  3. Vaughan, C. K., Gohlke, U., Sobott, F., Good, V. M., Ali, M. M., Prodromou, C., Robinson, C. V., Saibil, H. R. and Pearl, L. H. (2006). Structure of an Hsp90-Cdc37-Cdk4 complex. Mol Cell 23(5): 697-707.
  4. Verba, K. A., Wang, R. Y., Arakawa, A., Liu, Y., Shirouzu, M., Yokoyama, S. and Agard, D. A. (2016). Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase. Science 352(6293): 1542-1547.

简介

Hsp90,其伴侣伴侣Cdc37和激酶之间的相互作用已经在三十多年的生物化学研究中被证明在酵母与人类的生物体内在功能上是重要的。 然而,形成一个稳定的结构研究复合物是难以捉摸的。 在该方案中,我们描述了利用病毒2A序列以相似水平滴定三种蛋白质的来自酿酒酵母的Hsp90-Cdc37-Cdk4激酶蛋白复合物的表达和纯化。
【背景】Hsp90分子伴侣与其客体激酶之间的稳定形成复合物已经被证明在体外是难治性的。以前的工作表明,Hsp90的共伴伴Cdc37与昆虫Sf9细胞中的客体激酶的过表达导致Sf9 Hsp90,外源Cdc37和外源激酶(Vaughan等人,2006)之间的稳定复合物。然而,昆虫细胞培养需要特殊的设备,比其他研究较好的表达系统(如细菌和酵母)要难以进行遗传操作,并且显着较慢地生长和克隆。上述蛋白质在E中的共表达。大肠杆菌不产生可溶性激酶/稳定复合物。我们认为,酿酒酵母将具有必要的机制来帮助折叠和促进复合物的形成,并试图通过共同表达这些蛋白质来产生人Hsp90β,人Cdc37和人Cdk4激酶之间的复合物, S上。酵母。为了获得三种蛋白质的化学计量表达,我们利用病毒2A肽,其允许三个蛋白质在一个mRNA上转录,随后在翻译阶段切割。该系统已被用于人类细胞系和兔网状细胞(Kim等人,2011; Minskaia和Ryan,2013),但据我们所知,这是第一次利用2A病毒表达肽 S上。酵母。

关键字:HSP90, Cdc37, Cdk4, 伴侣, 激酶, 2A肽, 酵母蛋白表达

材料和试剂

    生成共表达式构造
    1. 100 x 15 mm培养皿(Fisher Scientific,目录号:FB0875713)
    2. 15ml培养管(Corning,Falcon ®,目录号:352051)
    3. 1.5ml微量离心管(Fisher Scientific,目录号:05-408-129)
    4. 0.2μm过滤器
    5. 83nu载体(从Arkin实验室获得)
    6. GeneArt Strings DNA(Thermo Fisher Scientific)
    7. NEB Builder装配工具(在线: http://nebuilder.neb.com/
    8. 吉布森装配克隆试剂盒(New England Biolabs,目录号:E5510S)

    9. PCR清洁工具包(Promega,目录号:A9281)
    10. Miniprep Kit(Omega Bio-tek,目录号:D6942-02)
    11. Q5位点定向诱变试剂盒(New England Biolabs,目录号:E0554S)
    12. Bacto-tryptone(BD,Bacto TM ,目录号:211705)
    13. 碳青霉素(Gold Bio,目录号:C-103-100)
    14. 酵母提取物(BD,Bacto TM ,目录号:212750)
    15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-10KG)
    16. 琼脂(BD,Difco TM ,目录号:281210)
    17. LB(见配方)
    18. LB琼脂(见食谱)

  1. Hsp90 / Cdc37 / Cdk4的表达
    1. 一次性分光光度计电池(Agilent Technologies,目录号:6610018700)
    2. 酵母菌JEL1菌株(MAT-alpha,leu2 trp1 ura3-52 prb1-1122 pep4-3 deltahis3 :: PGAL10-GAL4)。乌克兰西南部卢克莱斯的礼物
    3. 冷冻EZ酵母转化II试剂盒(ZYMO研究,目录号:T2001)
    4. 半乳糖(Sigma-Aldrich,目录号:G0625)
    5. 酵母氮基(YNB)(BD,Difco TM,目录号:291940)
    6. 葡萄糖(Sigma-Aldrich,目录号:G8270)
    7. CSM-His氨基酸混合物(MP Biomedicals,目录号:4510-312)
    8. 蛋白胨(BD,Bacto TM ,目录号:211820)
    9. 酵母提取物(BD,Bacto TM ,目录号:212750)
    10. DL-乳酸钠溶液(Sigma-Aldrich,目录号:L1375)
    11. 甘油(Sigma-Aldrich,目录号:G5516)
    12. SD-His(见食谱)
    13. YPGL媒体(见配方)

  2. Hsp90 / Cdc37 / Cdk4复合物的纯化
    1. 具有16G针头的30ml注射器(BD,目录号:309650)(BD,目录号:305196)
    2. 50ml锥形管(Corning,目录号:352070)
    3. 浓缩器,15ml,30kDa截止值(EMD Millipore,目录号:UFC903096)
    4. 透析管,10kDa截止值(Thermo Fisher Scientific,Thermo Scientific TM,目录号:68100)
    5. 12 x 75 mm管用于收集馏分(Fisher Scientific,目录号:14-959-16)
    6. FLAG肽(GenScript,目录号:RP10586)
    7. 蛋白酶抑制剂,不含EDTA(Roche Diagnostics,目录号:11873580001)
    8. Ni-NTA超流珠(QIAGEN,目录号:30450)
    9. 抗FLAG M2磁珠(Sigma-Aldrich,目录号:M8823)
    10. 在实验室中制备的TEV蛋白酶为10mg / ml,与(Sigma-Aldrich,目录号:T4455)相同,
    11. 液氮
    12. 螺栓4-12%Bis-Tris Plus凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:NW04120BOX)
    13. NuPAGE LDS样品缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0007)
    14. NuPAGE MOPS SDS运行缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0001)
    15. Trizma碱(Sigma-Aldrich,目录号:T1503)
    16. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-10KG)
    17. 咪唑(Sigma-Aldrich,目录号:I2399)
    18. 氯化镁(MgCl 2),1M储备(Sigma-Aldrich,目录号:63069)
    19. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    20. 钼酸钠(NaMoO 4)(Sigma-Aldrich,目录号:243655)
    21. 二硫苏糖醇(DTT)(Gold Bio,目录号:DTT100)
    22. 裂解缓冲液(见配方)
    23. 透析缓冲液(见配方)
    24. 凝胶过滤缓冲液(见配方)

设备

  1. 生成共表达式构造
    1. PCR机(如Biorad DNA Engine BG96TC)
    2. 37℃培养摇床(如新不伦瑞克Innova 43系列)
    3. 4°C和-20°C冰箱(任何品牌)
    4. 42℃的热块(如Thomas Scientific 3661044)

  2. Hsp90 / Cdc37 / Cdk4的表达
    1. 2.5L烧瓶(Ultra Yield)(Thomson Instrument,目录号:931136-B)
    2. 高压灭菌器
    3. 使用1升瓶子(如Beckman Coulter,型号:Avanti J20系列)的具有3,000 x g的离心机
    4. 30℃的培养振荡器,能够在200rpm下振荡250ml和1L烧瓶(如Eppendorf,New Brunswick ,型号:Innova 4200系列)
    5. 紫外可见分光光度计(Agilent Technologies,型号:Agilent 8453)

  3. Hsp90 / Cdc37 / Cdk4复合物的纯化
    1. EmulsiFlex-C3(Avestin,型号:EmulsiFlex-C3)
    2. 离心机(如Beckman Coulter,型号:Avanti ® J20系列)
    3. 高速离心管(如Beckman Coulter,目录号:357002)具有30,000 x g的能力)
    4. Rocker(如Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88880019)
    5. MonoQ 10 / 100GL专栏(GE Healthcare,目录号:17-5167-01)
    6. HiLoad 16/600 Superdex 200pg色谱柱(GE Healthcare,目录号:28989335)
    7. 玻璃重力柱(Bio-Rad Laboratories,目录号:7372512)
    8. ÄKTApurifier系统(GE Healthcare,型号:ÄKTApurifier系统)
    9. -80°C冰箱(如Eppendorf,New Brunswick TM ,型号:U570 ULT)
    10. 烧杯尺寸分类
    11. 磁性支架(如EMD Millipore,目录号:LSKMAGS15)
    12. 迷你凝胶罐(Thermo Fisher Scientific,目录号:A25977)
    13. PowerEase 500电源(Thermo Fisher Scientific,型号:PowerEase ® 500)
    14. 液氮烧瓶(如Thermo Fisher Scientific,Thermo Scientific TM,目录号:2129)
    15. Spatulas(Cole-Parmer,目录号:UX-06369-16)

程序

  1. 生成共表达式构造
    1. 为了共表达人类Hsp90β,人类Cdc37和人类Cdk4,我们利用病毒2A肽。这样我们可以构建一个单一的质粒,其中有三个蛋白质。 (参见图1)我们使用的确切的2A序列(称为P2A)来自猪Teschovirus-1,并且是GSGATNFSLLKQAGDVEENPGP。得到的结构是这样的:hCdc37-TEVsite-P2A-hHsp90β-TEVsite-FLAG-P2A-hCdk4-TEVsite-HisTag。将其插入hCdc37和hHsp90之间的核苷酸顺序为:CAGAACCTGTACTTTCAGGGCGGATCCGGTGCCACCAACTTTAGCTTGTTGAAGCAGGCTGGAGACGTGGAGGAAAATCCTGGACCC,hHsp90和hCdk4之间是:CAAAACTTATACTTCCAGGGTGACTATAAGGACGATGACGACAAAGGCTCCGGTGCCACCAACTTCTCATTATTGAAACAGGCCGGTGATGTAGAGGAAAATCCAGGACCT,和hCdk4是后:CAGAATCTGTATTTTCAGGGACATCATCACCATCACCATTAATGA。上述完整序列是密码子优化的(手工检查蛋白质之间的区域是不同的,以方便克隆)。有关完整插入,请参阅附录序列。


      图1.使用Hsp90,Cdc37和Cdk4设计和克隆构建体

    2. 然后将该序列分成两部分(核苷酸编号为2,212),并将两部分序列作为Thermo Fisher Scientific的GeneArt Strings DNA。使用NEBuilder,引物设计用于扩增83nu载体和每个GeneArt Strings DNA,以使用Gibson Assembly Cloning Kit克隆它们。
    3. 83nu载体,并使用PCR机器扩增每个GeneArt Strings DNA,然后在37℃下用脯氨酸在37℃下消化过夜(O / N)(〜16小时),PCR清洗,组装使用吉布森装配克隆试剂盒并转化到大肠杆菌TOP10细胞,该试剂盒在37℃下在LB-羧苄青霉素平板上铺板并生长O / N。
    4. 第二天,挑选10个菌落,并开始一个5ml培养的LB和100mg / L的羧苄青霉素和每个菌落。
    5. 在37℃振荡24小时后,用小剂量试剂盒提取质粒DNA,送出测序(通过整个基因平铺每500bp)。在我们的例子中,由于合成错误,最好的克隆有一个突变,使用Q5 Site Directed Mutagenesis Kit和其中的方案进行了修正。
    6. 将最终的构建物测序并储存在-20℃的蒸馏的无核酸酶的水中以备将来使用。 4°C冰箱用于短期储存DNA(不到一周)。

  2. Hsp90 / Cdc37 / Cdk4的表达
    1. 使用Frozen-EZ酵母转化II试剂盒方案将该质粒转化到JEL1(MAT-α,leu2 trp1 ura3-52nprb1-1122 pep4-3 deltahis3 :: PGAL10-GAL4)酵母菌株中并接种于SD-His盘。
    2. 3天后,挑取菌落并灌注到100ml的SD-His培养基中,在250ml烧瓶中,并以30℃振荡200rpm振荡生长。
    3. 第二天,将6×1L烧瓶的YPGL培养基(高压灭菌)接种10ml各O / N培养物。
    4. 在30℃和200rpm(在分光光度计上测量的光密度[OD]为1)约24小时后,加入终浓度为2%w / v的粉末状半乳糖以诱导蛋白质表达。
    5. 培养物在3,000xg生长6小时后沉淀20分钟。
    6. 将丸粒舀起并在-80℃冷冻。

  3. Hsp90 / Cdc37 / Cdk4复合物的纯化
    1. 首先用抹刀将细胞沉淀物重新悬浮在蛋白酶抑制剂的裂解缓冲液中,然后将样品通过16 G注射器针头(总共约为160ml,6L酵母培养物)。
    2. 然后通过EmulsiFlex-C3机器在25,000 psi下通过裂解物,5个完整通过,按照标准制造商的程序进行裂解。
    3. 通过以30,000×g离心30分钟清除裂解物。
    4. 使用玻璃重力柱在室温下平衡5ml体积的具有20床体积(100ml)裂解缓冲液的Ni-NTA珠粒。将离心旋转的上清液加入到珠粒中,然后在4℃下在封盖的玻璃重力柱中摇动1小时。
    5. 孵育1小时后,用20个床体积的裂解缓冲液洗涤珠子,然后用两个步骤的玻璃重力柱洗脱至10个床体积的裂解缓冲液+ 500mM咪唑。
    6. 然后将所得洗脱液与预平衡(10床体积的裂解缓冲液,使用磁性架)的抗FLAG M2磁珠(1ml床体积)在50ml塑料锥形管中在4℃下摇摆1小时,摇动。
    7. 用10个床体积的裂解缓冲液洗涤珠子,使用磁性支架和15ml管,并用3个床体积的具有75μg/ ml FLAG肽的裂解缓冲液洗脱样品,每次两次,25分钟。
    8. 将TEV蛋白酶加入到洗脱液(2.5mg)中,并使用透析管向4L透析缓冲液O / N透析样品。
    9. 用没有NaCl的透析缓冲液稀释样品1:1,并在ÄKTApurifier系统上负载到预平衡的MonoQ 10 / 100GL柱上。
    10. 洗去未结合的样品(约3 CV [柱体积])后,梯度运行至1M NaCl(超过20 CV),分馏以洗脱结合的复合物(以约25%的电导率脱落)。
    11. 将级分合并,浓缩,然后装入在凝胶过滤缓冲液中预平衡的HiLoad 16/600 Superdex 200pg柱上。
    12. 将峰分数(约0.5CV)合并,浓缩,在液氮中快速冷冻并储存在-80℃。在纯化期间的各个点(参见图2),等分试样在SDS-PAGE凝胶上运行(图2)。


      图2.来自酵母的Hsp90-Cdc37-Cdk4纯化的SDS-PAGE凝胶。A.纯化过程中,SDS-PAGE凝胶在不同步骤显示等分试样。细胞裂解物 - 纯化后的步骤C2,步骤C3的上清液 - 上清液,His珠粒在步骤C5流过 - 流过,His珠洗脱洗脱在步骤C5,Flag珠粒流过标志珠子流过步骤C6,在步骤7中标记珠洗脱洗脱。B.两个凝胶是MonoQ 10 / 100GL色谱柱和大小排除柱的分数。

数据分析

随后用低温电子显微镜(cryoEM)准备样品,然后进行数据采集和重建。在Verba等人,2016年中详细描述了用于这些步骤的协议。

笔记

  1. 升级纯化Genscript Anti-DYKDDDDK G1亲和树脂(L00432)也成功用于玻璃重力柱而不是Anti-FLAG M2 Magentic Beads。使用相同数量的珠子/洗涤/洗脱缓冲液的比例。
  2. 该方案也成功应用于Hsp90 / Cdc37 / Her2复合物的表达和纯化。
  3. 单独的激酶的表达,没有Hsp90 / Cdc37产生可溶性活性激酶。

食谱

  1. LB
    10克细菌胰蛋白胨
    5克酵母提取物
    10克NaCl
    在1升MilliQ水中 高压灭菌器
    如果加入抗生素,如羧苄青霉素,将50毫克/毫升储备液(0.2微米无菌过滤)加到冷却至低于50℃的LB中,终浓度为50毫克/升。
  2. LB琼脂
    如上所述,15克琼脂
    如果加入抗生素,如羧苄青霉素,将50毫克/毫升储备液(0.2微米无菌过滤)加到冷却至低于50℃的LB中,终浓度为50毫克/升。
  3. SD-His
    6.7克酵母氮碱基
    5g葡萄糖
    0.77克CSM-His氨基酸混合物
    在1升MilliQ水中(或100毫升为10倍)
    无菌过滤器
    对于平板按上述方法进行,但每升15克琼脂/
  4. YPGL媒体
    2%蛋白胨
    1%酵母提取物
    3%甘油
    2%乳酸盐
    在1升去离子水中 高压灭菌器
  5. 裂解缓冲液
    20 mM Tris pH 7.5
    150 mM NaCl
    20 mM咪唑
    10mM MgCl 2
    10 mM KCl
    20mM NaMoO 4
  6. 透析缓冲液
    20 mM Tris pH 7.5
    100 mM NaCl
    10mM MgCl 2
    10 mM KCl
    20mM NaMoO 4
  7. 凝胶过滤缓冲液
    20 mM Tris pH 7.5
    150 mM NaCl
    10 mM KCl
    20mM NaMoO 4
    1 mM DTT
将级分合并,浓缩,然后装入凝胶过滤缓冲液中预平衡的HiLoad 16/600 Superdex 200pg柱上。
  • 将峰分数(约0.5CV)合并,浓缩,在液氮中快速冷冻并储存在-80℃。在纯化期间的各个点(参见图2),等分试样在SDS-PAGE凝胶上运行(图2)

    图2.来自酵母的Hsp90-Cdc37-Cdk4纯化的SDS-PAGE凝胶.A。纯化过程中,SDS-PAGE凝胶在不同步骤显示等分试样。细胞裂解物 - 纯化后的步骤C2,步骤C3的上清液 - 他的珠粒在步骤C5流过 - 流过,他的珠洗脱洗在步骤C5,旗珠粒流过标志珠子流过步骤C6,在步骤7中标记珠洗脱洗脱.B。两个凝胶是MonoQ 10 / 100GL色谱柱和大小排除柱的分数。
  • 数据分析

    随后用低温电子显微镜(cryoEM)准备样品,然后进行数据采集和重建。在维巴等人,2016年中详细描述了用于这些步骤的协议。

    笔记

    1. 升级纯化Genscript Anti-DYKDDDDK G1亲和树脂(L00432)也成功用于玻璃重力柱而不是Anti-FLAG M2 Magentic Beads。使用相同数量的珠子/洗涤/洗脱缓冲液的比例。
    2. 该方案也成功应用于Hsp90 / Cdc37 / Her2复合物的表达和纯化。
    3. 单独的激酶的表达,没有Hsp90 / Cdc37产生可溶性活性激酶。

    食谱

    1. LB
      10克细菌胰蛋白胨
      5克酵母提取物
      10克NaCl
      在1升的MilliQ水中 高压灭菌器
      如果加入抗生素,如羧苄青霉素,将50毫克/毫升储备液(0.2微米无菌过滤)加到冷却至低于50℃的LB中,终浓度为50毫克/升。
    2. LB琼脂
      如上所述,15克琼脂
      如果加入抗生素,如羧苄青霉素,将50毫克/毫升储备液(0.2微米无菌过滤)加到冷却至低于50℃的LB中,终浓度为50毫克/升。
    3. SD-His
      6.7克酵母氮碱基
      5g葡萄糖
      0.77克CSM-His氨基酸混合物
      在1升MilliQ水中(或100毫升为10倍)
      无菌过滤器
      对于平板按上述方法进行,但每升15克琼脂/
    4. YPGL媒体
      2%蛋白胨
      1%酵母提取物
      3%甘油
      2%乳酸盐
      在1升去离子水中 高压灭菌器
    5. 裂解缓冲液
      20 mM Tris pH 7.5
      150 mM NaCl
      20 mM咪唑
      10mM MgCl 2
      10 mM KCl
      20mM NaMoO 4
    6. 透析缓冲液
      20 mM Tris pH 7.5
      100 mM NaCl
      10mM MgCl 2
      10 mM KCl
      20mM NaMoO 4
    7. 凝胶过滤缓冲液
      20 mM Tris pH 7.5
      150 mM NaCl
      10 mM KCl
      20mM NaMoO 4
      1 mM DTT

    致谢

    感谢秋叶彦(横山实验室)分享昆虫细胞纯化方案,酵母表达载体姚凡(Arkin Lab)和D.A.A.实验室成员进行有益的讨论,特别是Miguel Betegon提出使用病毒2A肽和Michelle Moritz进行酵母表达菌株的建议。 PSI-Biology授予U01 GM098254(至DAA),AACR-BCRF授予218084转化乳腺癌研究(至DAA),卡巴拉家庭礼物(DAA),HHMI国际学生研究奖学金(至KV )和霍华德休斯医学研究所(达达)。

    参考

    1. Kim,JH,Lee,SR,Li,LH,Park,HJ,Park,JH,Lee,KY,Kim,MK,Shin,BA和Choi,SY(2011)。< a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/21602908”target =“_ blank”>在人类细胞系,斑马鱼和小鼠中源自猪teschovirus-1的2A肽的高切割效率。 e 6(4):e18556。
    2. Minskaia,E.和Ryan,MD(2013)。使用FMDV 2A的蛋白质共表达:“接头”残基的作用。2013年:291730.Biomed Res Int。
    3. Vaughan,CK,Gohlke,U.,Sobott,F.,Good,VM,Ali,MM,Prodromou,C.,Robinson,CV,Saibil,HR and Pearl,LH(2006)。 Hsp90-Cdc37-Cdk4复合物的结构。细胞 23(5):697-707。
    4. Verba,KA,Wang,RY,Arakawa,A.,Liu,Y.,Shirouzu,M.,Yokoyama,S.and Agard,DA(2016)。  Hsp90-Cdc37-Cdk4的原子结构揭示Hsp90捕获和稳定未折叠的激酶。科学 352(6293):1542-1547。
    • English
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    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Verba, K. A. and Agard, D. A. (2017). Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae. Bio-protocol 7(19): e2563. DOI: 10.21769/BioProtoc.2563.
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