Hsp90-Cdc37-Cdk4激酶复合物在酿酒酵母中的表达和纯化

Kliment A. Verba; David A. Agard

doi:10.21769/BioProtoc.2563

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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

KV Kliment A. Verba

DA David A. Agard email

发布: 2017年10月05日第7卷第19期 DOI: 10.21769/BioProtoc.2563 浏览次数: 8493

评审: Arsalan DaudiYann Simon GallotAnonymous reviewer(s)

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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)

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

100 x 15 mm Petri dishes (Fisher Scientific, catalog number: FB0875713 )
15 ml culture tubes (Corning, Falcon^®, catalog number: 352051 )
1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129 )
0.2 μm filter
83nu vector (obtained from Arkin lab)
GeneArt Strings DNA (Thermo Fisher Scientific)
NEB Builder Assembly Tool (Online: http://nebuilder.neb.com/)
Gibson Assembly Cloning Kit (New England Biolabs, catalog number: E5510S )
DpnI
PCR Clean up Kit (Promega, catalog number: A9281 )
Miniprep Kit (Omega Bio-tek, catalog number: D6942-02 )
Q5 Site-Directed Mutagenesis Kit (New England Biolabs, catalog number: E0554S )
Bacto-tryptone (BD, Bacto^TM, catalog number: 211705 )
Carbenicillin (Gold Bio, catalog number: C-103-100 )
Yeast extract (BD, Bacto^TM, catalog number: 212750 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-10KG )
Agar (BD, Difco^TM, catalog number: 281210 )
LB (see Recipes)
LB agar (see Recipes)

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, Difco^TM, 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, Bacto^TM, catalog number: 211820 )
9. Yeast extract (BD, Bacto^TM, 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)
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 Scientific^TM, 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, Invitrogen^TM, catalog number: NW04120BOX )
13. NuPAGE LDS sample buffer (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0007 )
14. NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific, Invitrogen^TM, 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

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)
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 Brunswick^TM, model: Innova^®4200 series)
5. UV-Visible spectrophotometer (Agilent Technologies, model: Agilent 8453 )
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 Scientific^TM, 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 Brunswick^TM, 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 Scientific^TM, catalog number: 2129 )
15. Spatulas (Cole-Parmer, catalog number: UX-06369-16 )

Procedure

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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.