去NEDD化修饰的体外试验

Anna M. Köhler; Cindy Meister; Gerhard H. Braus

doi:10.21769/BioProtoc.1756

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Peer-reviewed

In vitro Deneddylation Assay

去NEDD化修饰的体外试验

Anna M. Köhler

Cindy Meister

Gerhard H. Braus email

发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1756 浏览次数: 9383

评审: Valentine V TrotterManuela RoggianiAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Jul 2015

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Abstract

Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western hybridization experiments using anti-CulA or anti-Nedd8 antibodies could show the ratio of neddylated vs. deneddylated CulA. Using the deneddylation assay, we could show that CsnE is the last subunit joining a 7-subunit pre-assembled CSN in vitro and only then CSN can perform cullin deneddylation by the metalloprotease subunit CsnE. This assay is a fast and non-expensive method, which visualizes enzyme activity for deneddylating proteins. It might be also useful for testing the activity of other isopeptidases removing post-translational modifications from substrates in Aspergillus nidulans (A. nidulans) or other organisms.

Keywords: Deneddylation (deneddylation)

COP9 signalosome (CSN) (COP9 signalosome（CSN）)

DEN1 (DEN1)

Western hybridization (Western杂交)

Nedd8 (NEDD8)

Materials and Reagents

Recombinant protein overexpression and protein purification
1. 1 ml cuvettes 10 x 10 x 45 mm (Sarstedt AG, catalog number: 67.742 )
2. Chromatography columns [e.g. GSTrap (GE Healthcare, catalog number: 17-5130-01 ), HisTrap (GE Healthcare, catalog number: 17-5255-01 ), Superdex 200 HR 10/300 GL (GE Healthcare, catalog number: 17-5175-01 ), HiPrep 16/60 Sephacryl S-300 HR (GE Healthcare, catalog number: 17-1167-01 )]
3. Amicon Ultra centrifugal filters (Merck Millipore Corporation) molecular weight cut off (MWCO) depends on protein size [e.g. 30 kDa (Merck Millipore Corporation, MWCO, catalog number: UFC903024 )]
4. MicronSep filter paper, 0.22, 47 mm (Carl Roth GmbH + Co., catalog number: A009.1 )
5. Escherichia coli (E. coli) strains suitable for protein overexpression (e.g. Rosetta DE3) transformed with overexpression plasmid including protein sequence of interest with N- or C-terminal Glutathione-S-transferase- (GST-) or Histidine- (His-) tag
6. Tryptone/Peptone from Casein (Carl Roth GmbH + Co., catalog number: 8952.2 ) used as 1% solution
7. Yeast extract (Oxoid, catalog number: LP0021 ) used as 0.5% solution
8. Sodiumchloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2 ) used as 1% solution
9. Antibiotics according to the E. coli strains used
10. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth GmbH + Co., catalog number: 2316.4 ) used as 1 M solution, filter sterilized
11. 99.5% denatured Ethanol with 1% MEK (VWR International, catalog number: 85033.460 ) used as 20% solution diluted in ddH₂O, filter degassed
12. Imidazole
13. Glutathione
14. Lysogeny broth (LB) medium (see Recipes)
15. Lysis buffer (see Recipes)
16. Affinity buffer (see Recipes)
17. Elution buffer for HisTrap (see Recipes)
18. Elution buffer for GSTrap (see Recipes)
19. Size exclusion buffer (SEC) (see Recipes)
Preparation of protein crude extracts
1. Paper towels
2. Spores of Aspergillus nidulans strains
3. Miracloth (Merck Millipore Corporation, catalog number: 475855-1R )
4. Sodiumchloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2) used as 0.95 % solution
5. Liquid nitrogen (Air Liquid)
6. Minimal medium (see Recipes)
7. 50x AspA (see Recipes)
8. Trace elements (see Recipes)
9. B* buffer (see Recipes)
Deneddylation assay
1. Gel blotting paper (Schleicher & Schuell BioScience GmbH, catalog number: 10426694 )
2. Blotting membrane Amersham^TM Protran^TM 0.45 µm NC (GE Healthcare, catalog number: 10600002 )
3. Methanol (VWR International, catalog number: 20864.320 )
4. 2-Propanol HiPerSolv CHROMANORM (VWR International, catalog number: 20880.320 )
5. Page ruler prestained protein ladder (Thermo Fisher Scientific, catalog number: 26616 )
6. Acrylamid Rotiphorese^® Gel 30 (37, 5:1) (Carl Roth GmbH + Co., catalog number: 3029.1 )
7. Sucofin skimmed milk powder (TSI GmbH, catalog number: ST00227 )
8. α-CulA, α-Nedd8 antibodies (GenScript)
9. Monoclonal α-Tubulin antibody (Sigma-Aldrich, catalog number: T0926 )
10. α-Actin antibody (Novus Biologicals, catalog number: NB100-74340 )
11. α-goat rabbit secondary antibody (Thermo Fisher Scientific, Invitrogen^TM, catalog number: G21234 )
12. α-goat mouse secondary antibody (Jackson ImmunoResearch, catalog number: 115-035-003 )
13. p-coumaric acid (Sigma-Aldrich, catalog number: C9008-5 G ) used as 400 mM stock solution dissolved in DMSO (protect from light, store at -20 °C)
14. Luminol (Carl Roth GmbH + Co., catalog number: 4203.1 ) used as 250 mM stock solution dissolved in DMSO (protect from light, store at -20 °C)
15. 2-mercaptoethanol (Sigma-Aldrich, Fluka Chemika, catalog number: 63700 )
16. Sodium dodecyl sulfate (SDS) (Carl Roth GmbH + Co., catalog number: CN30.3 ) used as 7%, 0.1%, 1%, 0.2% solutions
17. Bromophenol blue (Sigma-Aldrich, Riedel-de Haën, catalog number: 32768 ) used as 0.3% solution
18. Ammoniumpersulfate (APS) (Sigma-Aldrich, Fluka Analytical, catalog number: 09913-100 G ) used as 10% solution
19. Tetramethylethylendiamine (TEMED) (Carl Roth GmbH + Co., catalog number: 2367.1 )
20. Tween-20 (Carl Roth GmbH + Co., catalog number: 9127.1 )
21. Tris-PUFFERAN^® (Carl Roth GmbH + Co., catalog number: 4855.3 ) used as 250 mM solution
22. Glycine (Carl Roth GmbH + Co., catalog number: 3187.3 ) used as 2.5 M solution
23. 30% Hydrogen peroxide (H₂O₂) (Merck Millipore Corporation, catalog number: 822287.2500 )
24. 3x SDS sample buffer (see Recipes)
25. 12% SDS gels (see Recipes)
26. 2x resolving buffer (see Recipes)
27. 2x stacking buffer (see Recipes)
28. 10x SDS running buffer (see Recipes)
29. 10x transfer buffer (see Recipes)
30. 10x TBST (Tris-Buffered Saline Tween 20) buffer (see Recipes)
31. Blocking solution (see Recipes)
32. Detection solution A (see Recipes)
33. Detection solution B (see Recipes)
Others (material and reagents used for more than one method)
1. Micro tube 1.5 ml (Sarstedt AG, catalog number: 72.690.001 )
2. Micro tube 2.0 ml (Sarstedt AG, catalog number: 72.691 )
3. Tube 115 x 28 mm, PP (Sarstedt AG, catalog number: 62.559.001 )
4. 5 ml test tube
5. 2 L flasks
6. PPCO centrifuge bottles (Slaughte, Nalgene, catalog number: 525-2316 )
7. Oak-Ridge centrifugation tubes, 50 ml (Carl Roth GmbH + Co., catalog number: PK03.1 )
8. 300 ml baffled flasks
9. Funnels
10. Crushed ice
11. 1, 4- Dithiothreitol (DTT) (Carl Roth GmbH + Co., catalog number: 6908.2 ) used as 1 M solution
12. trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane, E-64 protease inhibitor (Sigma-Aldrich, catalog number: E3132 ) used as 1 M stock solution
13. Phenylmethylsulfonyl fluoride (PMSF) (Carl Roth GmbH + Co., catalog number: 6367.3 ) used as 100 mM solution in Isopropanol
14. Double destilled water (ddH₂O)
15. Millipore water
16. Sodium chloride (NaCl) (Carl Roth GmbH + Co., catalog number: 9265.2) used as 2 M solution
17. Tris- hydrochloride (Tris-HCl) (Carl Roth GmbH + Co., catalog number: 9090.3 )
  Note: Prepare a series of Tris-HCl solution with different concentrations and pH including 1 M solutions with pH 6.8, 7.5, 8.0 and pH 8.8; 0.75 M solution withpH 8.8; 100 mM solution with pH 8.0.
18. Ethylenediaminetetraacetic acid (EDTA) (Carl Roth GmbH + Co.) used as 0.5 mM solution
19. Nonoxinol-40 (NP-40) (Affymetrix, USB Corporation, catalog number: 19628 )
20. Glycerol (Carl Roth GmbH + Co., catalog number: 3783.1 )
21. Complete protease inhibitor cocktail (Roche Diagnostics, catalog number: 11873580001 ) (see Recipes)

Equipment

Biowave DNA Llife Science Spectrophotometer (Biochrom WPA, catalog number: 80-3004-70 )
Centrifuge Sorvall RC3B Plus (Thermo Fisher Scientific)
Centrifuge Sorvall RC5B Plus (Thermo Fisher Scientific)
Rotor Sorvall SS34 (Thermo Fisher Scientific)
ÄKTAexplorer 10 (GE Healthcare, catalog number: 18-1300-00 )
pH meter pH526 (Sigma-Aldrich, model: WTW )
Filter and degassing system (Satorius)
Vacuum pump (KNF NEUBERGER, catalog number: 2.03360238 )
Sonicator Sonoplus HD2070 (Bandelin)
Superloop 1/16’’ (GE Healthcare, catalog number: 18-1113-82 )
Sample loop 5 ml (GE Healthcare, catalog number: 18-1140-53 )
Mixer Mill MM 400 (Retsch)
Vortex Genie 2 (Scientific Industries)
Pipettes (1-5,000 µl) (Gilson)
Centrifuge Biofuge fresco (Heraeus Instruments GmbH)
Nanodrop Spectrophotometer (VWR International, Peqlab)
Mini-PROTEAN^® Tetra Cell Casting Module (Bio-Rad Laboratories, catalog number: 1658008 )
Buffer Tank and Lid (Bio-Rad Laboratories, catalog number: 1658040 )
Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 1703935 )
Autoradiography casette
FUSION SL-4 3500.WL (Chemiluminescence System) (PEQLAB)
CCD camera FUSION 4 MP (VWR International, Peqlab)
Orbital Shaker 3015 (GFL)
Shaker (INFORS AG)
Heating block (Gebr. Liebisch GmbH & Co. KG Labortechnik)