Overview
This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting technique.
Protocol
1. Preparation of Baculovirus Expression Constructs
- Purify SUMO E3 ligase K-bZIP by cloning the cDNA of K-bZIP into a dual expression baculovirus vector (see Table of Materials) with an N-terminal epitope tag. We have had success using an octapeptide tag (indicated throughout the protocol as vector-tag-K-bZIP).
NOTE: The DNA template for K-bZIP polymerase chain reaction (PCR) cloning is cDNA reverse transcribed from RNA isolated from TREx F3H3-K-Rta BCBL-1 cells following doxycycline treatment.- Transfer the full-length K-bZIP cDNA into the dual expression vector with CpoI cloning sites. CpoI digests the PCR product and 1 µg of the dual expression vector at 37 °C for 3 h.
- Separate and extract the digested DNA following electrophoresis on a 0.8% agarose gel. Ligate the insert (K-bZIP) and vector by T4 DNA ligase at 16 °C for 30 min according to the manufacturer's instructions (see Table of Materials).
- Add 5 µL of ligation DNA to 50 µL competent E. coli cells "A" (see Table of Materials) in a 1.5 mL tube. Put on ice for 30 min. Then, incubate the 1.5 mL tube in a 42 °C water bath for 45 s and immediately put the tube on ice for 3 min.
- Add 600 µL S.O.C. medium (0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 20 mM MgSO4, and 4% (w/v) glucose) into the tube and incubate at 37 °C for 1 h. Then, centrifuge the tube at room temperature for 3 min at 600 x g.
- Remove supernatant, resuspend pellet with 60 µL Luria-Bertani medium (LB; 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 85 mM NaCl) and spread on LB agar plates containing 100 µg/mL ampicillin. Incubate the agar plate at 37 °C for 16 h.
- Select 1 colony to inoculate in 5 mL LB medium containing 100 µg/mL ampicillin and culture at 37 °C for 16 h with 250 rpm shaking. Then, extract the plasmid, vector-tag-K-bZIP, by a plasmid extraction kit according to the manufacturer's instructions (see Table of Materials).
- Generate the recombinant bacmid harboring the tag-K-bZIP by the transformation of the vector-tag-K-bZIP into competent E. coli cells "B" (see Table of Materials).
- Mix 0.2 µg vector-tag-K-bZIP plasmid and 100 µL competent E. coli cells "B" in a 1.5 mL tube and put on ice for 30 min. Incubate the tube in a 42 °C water bath for 45 s and immediately put the tube on ice for 3 min.
- Add 900 µL of S.O.C. medium into the tube and incubate at 37 °C for 4 h with rotation at 50 rpm. Next, take 10 µL of the medium, and add an additional 50 µL of S.O.C medium to spread on LB agar plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL galactosidase substrate, and 40 µg/mL isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubate the plates for 48 h at 37 °C.
- Inoculate one white colony in a 5 mL LB medium containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, and 10 µg/mL tetracycline. Incubate at 37 °C with 250 rpm shaking for 16 h.
- Isolate the recombinant bacmid DNA harboring the tag-K-bZIP, quantify, and re-suspend at a concentration of 1 µg/µL.
- Generate recombinant baculoviruses expressing tag-K-bZIP by transfecting 1 µg of recombinant bacmid DNA containing tag-K-bZIP into Sf9 cells in one well of a 6-well plate.
- Seed 2 x 106 Sf9 cells (see Table of Materials) in 2 mL Grace's Insect Medium supplied with 10% fetal bovine serum (FBS) and 1% gentamicin in each well of a 6-well plate, then incubate at 27 °C for 1 h.
- Maintain a log phase culture of Sf9 cells in Grace's Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C (see Table of Materials) at 27 °C in orbital suspension at 140 rpm. Count cells using a hemocytometer and trypan blue staining; cell viability should exceed 95%. Maintain cells using a subcultivation ratio of 1:3 every 2-3 days (minimal density ~0.5 x 106 cells/mL).
- Add 2 µL bacmid DNA (1 µg/µL) and 98 µL reduced serum media (see Table of Materials) into a 2 mL tube. Then, add 4 µL transfection reagent (see Table of Materials) to the tube and mix well. Incubate at room temperature for 15 min.
- Add this transfection mixture (104 µL) into each well, and incubate at 27 °C for 12-16 h.
- Remove exhausted media with gentle aspiration, and replace with 2 mL of fresh Grace's Insect Medium supplied with 10% FBS and 1% gentamicin. The Sf9 cells adhere loosely to the plate surface and will not be disturbed with gentle aspiration.
- Collect recombinant baculovirus after transfection and amplify baculovirus to obtain high-titer stocks (P1) for further experiments.
- 72 h after changing the medium, scrape the Sf9 cells using a sterile cell lifter, collect the supernatant from each individual well in a 1.5 mL tube, and vortex for 10 s.
- Centrifuge at 4 °C for 3 min at 150 x g. Collect supernatant as P0 baculovirus and aliquot in 1.5 mL tubes (1 mL baculovirus in each tube). Store at -80 °C.
- Seed 1 x 107 Sf9 cells in 10 mL Grace's Insect Medium supplied with 10% FBS and 1% gentamicin in a 10 cm Petri dish and incubate at 27 °C for 1 h.
- For amplification of recombinant baculoviruses expressing tag-K-bZIP, add 0.5 mL P0 baculovirus to the seeded Sf9 cells and incubate at 27 °C for 48 h.
- Collect supernatant as P1 baculovirus in a 15 mL tube and store at 4 °C for up to 1 month.
2. Purification of K-bZIP
- Maintain a log phase culture of Sf9 cells in Grace's Insect Medium supplied with 10% FBS, 1% gentamicin, and 1% detergent C at 27 °C in orbital suspension at 140 rpm as described in step 1.3.2.
- On the day of transduction, add 1 mL of baculovirus-containing supernatant (P1) into 250 mL of Sf9 cells with a density of 2 x 106 cells/mL.
- Incubate Sf9 cells for 72 h at 27 °C on an orbital shaker with 140 rpm shaking, then collect by centrifugation at 2,740 x g for 15 min at 4 °C.
- Remove supernatant and lyse cell pellets in 10 mL lysis buffer (20 mM HEPES pH 7.9, 0.5 M NaCl, 1% detergent A (see Table of Materials), 2% glycerol, protease inhibitor cocktail) and rotate at 50 rpm using a suspension mixer at 4 °C for 30 min.
- Centrifuge cell lysate at 15,000 x g for 15 min to collect the supernatant.
- To purify tag-K-bZIP, incubate the cell lysates (10 mL) with 50 µL of antibody-tagged magnetic beads in 14 mL polypropylene tubes, and rotate at 50 rpm for 3 h at 4 °C using a suspension mixer. Following protein capture, pellet the beads at 800 x g centrifugation for 30 s at 4 °C. Remove most of the supernatant, re-suspend the beads in the residual volume of about 1 mL, and transfer to a 1.5 mL tube for washing.
- Wash the captured protein by placing the tube containing the beads on a magnetic stand, and remove the supernatant once the magnetic beads have fully adhered to the side of the tube.
- Wash the beads with lysis buffer four times.
- Add 1 mL lysis buffer into 1.5 mL tubes, and invert the tubes 10 times. Place the 1.5 mL tube on a magnetic stand and remove the supernatant as described in 2.7.
- Repeat the previous step another 3 times.
- Wash the beads with phosphate-buffered saline (PBS) once.
- Add 1 mL PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4) into 1.5 mL tubes, and invert the tubes 10 times.
- Put the 1.5 mL tube on a magnetic stand and then remove the supernatant.
- Elute tag-K-bZIP protein from the antibody-tagged magnetic beads by adding 100 µL of 150 µg/mL octapeptide diluted in PBS into a 1.5 mL tube. Rotate the tube for 10 min at 50 rpm by a suspension mixer at room temperature. Then, place the tube back on the magnetic stand and collect the K-bZIP-containing supernatant in a clean 1.5 mL tube.
- Analyze 1-5 µL purified tag-K-bZIP protein by SDS-PAGE followed by Coomassie blue staining. Load 2 µg, 1 µg, and 0.5 µg samples of bovine serum albumin (BSA) on the same gel and use it for quantification with an image processing program, like ImageJ. Estimate the K-bZIP concentration by comparison to the BSA standards.
- The purified K-bZIP is now ready to be used in each SUMOylation assay. Dilute the K-bZIP in PBS to a final concentration of 100 ng/µL and store in small aliquots at -80 °C.
3. SUMOylation Assay
- Add 3 µL of 100 ng/µL of purified tag-K-bZIP into the master mix of each in vitro SUMOylation reaction. Components in master mix contain 1 µL protein buffer, 1x SUMOylation buffer, 0.5 µL p53 protein (0.5 mg/mL), 25 nM of E1 activating enzyme (stock conc.: 1 µM), 50 nM of E2 conjugating enzyme (stock conc.: 10 µM), and 250 nM each of the SUMO peptides (SUMO-1, SUMO-2, or SUMO-3; stock conc.: 5 µM) to a final volume of 17 µL.
- Mix the contents gently and incubate the reaction at 30 °C for 3 h. Stop the reaction by adding 20 µL of 2x SDS-PAGE loading buffer (100 mM Tris-HCL pH 6.8, 4% sodium dodecyl sulfate, 0.2% (w/v) bromophenol blue, 20% (v/v) glycerol, and 200 mM β-mercaptoethanol) and denature the samples at 95 °C for 5 min.
- Separate samples on SDS-PAGE, transfer the separated proteins onto PVDF membrane by using a semi-dry electrophoretic transfer apparatus, and analyze by Western blot using anti-p53 antibody.
- Set up the gel apparatus and load 20 µL of sample into gel wells.
- Run the 10% gel in a constant current at 80 V for about 120 min (until the dye band reaches the bottom of the gel). After completion of electrophoresis, turn off the power supply.
- Disconnect the gel apparatus and gel cassettes to take out the gel, and float the gel into semi-dry transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol) for 5 min.
- Take another container and soak the PVDF membrane in methanol for 1 min. Then, take PVDF out of methanol and put in semi-dry transfer buffer. Gently agitate the membrane for 5 min.
- Remove the safety cover of the semi-dry electrophoretic apparatus.
- Pre-wet filter paper, and prepare a gel sandwich on the bottom platinum anode as follows: filter paper, PVDF membrane, gel, and filter paper.
- Secure cathode plate and safety cover, then run blot at 15 V constant current for 90 min. Turn off the power supply, disconnect semi-dry apparatus, and take out the PVDF membrane.
- Block PVDF membrane with blocking buffer (5% non-fat milk in TBST buffer (137 mM NaCl2, 20 mM Tris-HCl, 0.1% detergent B (see Table of Materials), pH 7.6)) at room temperature for 1 h with 30 rpm shaking by orbital shaker.
- Hybridize the PVDF membrane with anti-p53 antibody diluted (1:1,000) in blocking buffer for 12-16 h at 4 °C with 30 rpm shaking using a suspension mixer.
- Take out the PVDF membrane into a container and put in TBST. Rinse PVDF membrane with TBST twice more.
- Soak the PVDF membrane in TBST for 30 min with 45 rpm shaking by orbital shaker.
- Hybridize the PVDF membrane with anti-rabbit antibody conjugated with horseradish peroxidase (HRP) diluted (1:4,000) in blocking buffer for 1 h at room temperature with 30 rpm shaking using a suspension mixer.
- Wash the PVDF membrane with TBST 3 times as described in step 3.4.10.
- Soak the PVDF membrane in TBST for 30 min with 45 rpm shaking using a suspension mixer. Remove TBST and add PBS to preserve the PVDF membrane at 4 °C for up to 12 h.
- Mix the enhanced chemiluminescent substrate (ECL substrate) reagent 1 and 2 (1:1) (see Table of Materials). Remove the PVDF membrane from the PBS, blot briefly with a paper towel or light-duty wiper to absorb excess moisture, and immediately add 400 µL of ECL reagent to the surface of each membrane for 3-5 min. Remove excess ECL reagent by briefly blotting, but do not allow the membrane to completely dry.
- Expose the blot using a luminescence imaging system or autoradiography film.
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Materials
Name | Company | Catalog Number | Comments |
pFastBac | Invitrogen | 10359-016 | dual expression baculovirus vector |
pCR2.1-TOPO vector | Invitrogen | PCR product vector | |
competent cell E. coli DH5α | Yeastern Biotech | FYE678-80VL | competent E. coli cells A |
E. coli DH10Bac | Invitrogen | 10359-016 | competent E. coli cells B |
FugeneHD | Roche | 4709705001 | transfection reagent |
Opti-MEM | Gibco | 31985062 | reduced serum media |
T4 Ligase | NEW England BioLabs | M0202S | |
CpoI (RsrII) | Thermo Scientific | ER0741 | |
Bluo-gal | Thermo Scientific | B1690 | galactosidase substrate |
IPTG | Sigma-Aldrich | I6758-1G | |
Grace’s Insect Medium | Gibco | 11605094 | |
Fetal Bovine Serum | Gibco | 10082147 | |
Gentamicin | Thermo Fisher | 15750060 | |
Ampicillin | Sigma-Aldrich | A9393-25G | |
Kanamycin | Sigma-Aldrich | K0254-20ML | |
gentamicin | Gibco | 15710-064 | |
tetracyclin | Sigma-Aldrich | 87128-25G | |
LB Broth | Merk | 1.10285.0500 | |
HEPES | Sigma-Aldrich | H4034-100G | |
NaCl | Sigma-Aldrich | S9888-5KG | |
KCl | Merk | 1.04936.1000 | |
Na2HPO4 | Sigma-Aldrich | S5136-500G | |
KH2PO4 | J.T.Baker | 3246-01 | |
sodium dodecyl sulfate | Merk | 1.13760.1000 | |
β-mercaptoethanol | Bio-Rad | 161-0710 | |
TRIS (Base) | J.T.Baker | 4109-06 | |
Non-fat milk | Fonterra | ||
glycerol | J.T.Baker | 2136-01 | |
Triton X-100 | Amresco | 0694-1L | detergent A |
Tween 20 | Amresco | 0777-500ML | detergent B |
Poloxamer 188 solution | Sigma-Aldrich | P5556-100ML | detergent C |
Protease Inhibitor Cocktail Tablet | Roche | 04 693 132 001 | |
3x Flag peptide | Sigma | F4799 | |
anti-FLAG m2 Magnetic beads | Sigma-Aldrich | M8823 | antibody-tagged magnetic beads |
SUMOlink SUMO-1 Kit | Active Motif | 40120 | standard SUMOylation protocol |
SUMOlink SUMO-2/3 Kit | Active Motif | 40220 | standard SUMOylation protocol |
QIAquick Gel Extraction Kit | QIAGEN | 28704 | |
QIAGEN Plasmid Mini Kit | QIAGEN | 12123 | plasmid extraction kit |
Polypropylene tubes | Falcon | 352059 | |
Petri Dish | Falcon | 351029 | |
Cell lifter | Corning | CNG3008 | |
Loading tip | Sorenson BioScience | 28480 | |
PVDF | PerkinElmer | NEF1002 | |
Blotting filter paper | Bio-Rad | 1703932 | |
Mini slab gel apparatus (Bio-Rad Mini Protean II Cell) | Bio-Rad | 1658001 EDU | |
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell | Bio-Rad | 1703940 | |
Pierce ECL Western Blotting | Thermo | 32106 | ECL reagent |
suspension mixer | Digisystem laboratory instruments Inc. | SM-3000 | |
orbital shaker | Kansin instruments Co. | OS701 | |
ImageQuant LAS 4000 | GE Healthcare | 28955810 | |
biomolecular imager | GE Healthcare | 28955810 | |
Sf9 | Thermo Scientific | B82501 | |
anti-p53 antibody | Cell Signaling | #9282 | |
anti-rabbit antibody | GE Healthcare | NA934-1ML |