2-1 Materials
2-1.1 Water
Water was distilled and deionized by Milli-RO PLUS 60 and Milli-QSP reagent water system (EMD Millipore, USA).
2-1.2 Chemicals
40% acrylamide/bis (29:1) solution Bio-Rad
95% industrial ethanol Uni-Onward (友和)
Absolute ethanol Merck KGaA
Acetic acid Sigma-Aldrich
Acetic acid, glacial J.T. Baker
Acetonitrile J.T. Baker
Agar AMRESCO
Agarose AMRESCO
Alexa Fluor 488® C5 maleimide dye Life Technologies
Ammonium sulfate AMRESCO
Ammonium persulfate Sigma-Aldrich
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Ampicillin sodium salt AMRESCO
Anti-ubiquitin antibody (rabbit) Sigma-Aldrich
ATP magnesium salt Sigma-Aldrich
BME (2-mercaptoethanol / β-mercaptoethanol) Sigma-Aldrich
Bis-Tris Sigma-Aldrich
Bromophenol blue AMRESCO
CelLytic B 10X reagent Sigma-Aldrich
Coomassie brilliant blue R-250 Sigma-Aldrich
DMSO (dimethyl sulfoxide) Sigma-Aldrich
DNase I Roche Applied Science
DpnI restriction enzyme New England Biolabs
DTT (dithiothreitol) AMRESCO
EDTA (ethylenediaminetetraacetic acid) J.T. Baker Formalin (formaldehyde solution 36.5 - 38%) Sigma-Aldrich
Glucose Sigma-Aldrich
Glycerol AMRESCO
Glycine AMRESCO
Goat anti-rabbit-IgG, HRP peroxidase conjugated EMD Millipore
HEPES J.T. Baker
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Magnesium chloride hexadydrate Merck KGaA
MES (2-(N-morpholino)ethanesulfonic acid) hydrate Sigma-Aldrich
Methanol alcohol anhydrous Macron Fine Chemicals
Nickel sulfate hexadydrate Riedel-de Haën
Potassium chloride J.T. Baker
Potassium phosphate monobasic Macron Fine Chemicals
Peptone from casein Merck KGaA / Bionovas
PfuUltra II fusion HS DNA polymerase Stratagene PMSF (phenylmethylsulfonyl fluoride) Sigma-Aldrich
SDS (sodium dodecyl sulfate) AMRESCO
Silver nitrate Sigma-Aldrich
Sodium acetate Sigma-Aldrich
Sodium carbonate Sigma-Aldrich
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Sodium chloride Sigma-Aldrich
Sodium hydroxide Sigma-Aldrich
Sodium phosphate monobasic Sigma-Aldrich
Sodium phosphate dibasic Sigma-Aldrich
Sodium thiosulfate Shimakyu’s Pure Chemicals
SUMO protease Ulp1 Invitrogen
T4 DNA ligase Roche Applied Science
T4 PNK (T4 polynucleotide kinase) New England Biolabs
TAE buffer (50X) AMRESCO
TCEP (tris(2-carboxyethyl)phosphine) Sigma-Aldrich
TEMED (tetramethylethylenediamine) AMRESCO
TFA (trifluoroacetic acid) Alfa Aesar
Tris (tris(hydroxymethyl)aminomethane) AMRESCO Tween-20 (Polyoxyethylene (20) sorbitan monolaurate) J.T. Baker
Ubiquitin Sigma-Aldrich
Ubiquitin activating enzyme (E1) human, recombinant Sigma-Aldrich Western chemiluminescent HRP substrate EMD Millipore
Yeast extract Merck KGaA / Bionovas
Zinc chloride Fluka
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2-2 Methods
2-2.1 Expression constructs and site-directed mutagenesis
We had plasmid containing ubiquitin gene in pET-11b vector. Because this construct had a 6-His tag on C-terminus of ubiquitin and could not be used in the ubiquitination assay, construct without 6-His tag were made from the tagged ones by PCR-based mutagenesis. Primers that changed the first histidine codon (CAT) on 6-His tag to a stop codon (TGA) were used. The primer sequences were listed below, with mutation sites underlined:
UbqDHis forward: 5’-CATTG AGGAT CCGGC TGCTA ACAAA-3’
UbqDHis reversed: 5’-ATGAT GATGA TGTCA ACCAC CTCTT AG-3’
Three Cys mutations M[C]Q (cysteine insertion between M1 and Q2), D39C (D39 is replaced by cysteine), and R72C (R72 is replaced by cysteine) were constructed by site-directed mutagenesis (D39C was constructed previously in our lab). In later experiments, seven lysine-to-arginine mutants of ubiquitin M[C]Q, namely, M[C]Q/K6R, M[C]Q/K11R, M[C]Q/K27R, M[C]Q/K29R, M[C]Q/K33R, M[C]Q/K48R, and M[C]Q/K63R, were also constructed by PCR-based method from the template, ubiquitin M[C]Q construct. The primer sequences used were listed below:
(mutation sites were underlined)
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M[C]Q/K6R forward M[C]Q/K6Rreversed
5’- AGGAC GTTAA CCGGT AAAAC CAT-3’
5’-GACGA AGATC TGGCA CATAT GTAT-3’
5’-AGGGC TAAAA TTCAA GACAA GGAAG G-3’
5’-AACGT TTTCG ATGGT ATCGG ATGG-3’
K29R forward K29R reversed
5’-CGAAT TCAAG ACAAG GAAGG CATTC C-3’
5’-AGCCT TAACG TTTTC GATGG TATCG G-3’
K33R forward
5’-CTGAA TGTTG TAATC AGACA GCGTT C-3’
After PCR amplification, the products were checked by electrophoresis in 1%
agarose gel and 1x TAE buffer (50x stock from AMRESCO, USA) under 100 volts for 30 minutes. The products were then treated with Dpn1 (New England Biolabs, USA) to digest parental plasmids, and purified by QIAquick PCR Purification Kit (QIAGEN, Germany). The purified plasmids were 5’-phosphorylated and ligated by T4
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polynucleotide kinase (New England Biolabs, USA) and T4 ligase (Roche Applied
Science, Germany), respectively. Ligated plasmids were first transformed into ECOSTM DH5α competent cells (Yeastern Biotech, Taiwan), cultured overnight on LB agar plate containing 100 μg/ml ampicillin. A few colonies on the plate were picked and
subcultured in LB medium. The amplified plasmids were extracted and purified by QIAprep Spin Miniprep Kit (QIAGEN, Germany). The clones with desired sequences were confirmed by DNA sequencing conducted in ABI PRISM® 96-capillary 3730 xl DNA Analyzer (Applied Biosystems, Life Technologies, USA) in DNA Sequencing Core Facility, Academia Sinica.
2-2.2 Small scale expression testing
Small scale expression tests were performed by transforming desired plasmid into E.
coli BL21 StarTM (DE3) competent cells (Invitrogen, Life Technologies, USA) on agar plate. Transformed cells were inoculated into LB medium with 100 μg/mL ampicillin and incubated overnight at 37°C, 250 rpm. 200 μL of overnight culture was subcultured into 5 ml LB/ampicillin medium and incubated for 3 hr. Then 5μL of 1M IPTG were added (with the final concentration of 1mM) and induced for 5 hr at 37°C, 250 rpm. The cell culture was centrifuged, then the cell pellet was collected and resuspeneded by lysis
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buffer (40 mM Tris, 150 mM NaCl, pH 8.0). SDS-PAGE sample buffer was added and the expression efficiency was checked by SDS-PAGE and Coomassie Blue R250 staining.
2-2.3 Large scale protein expression and purification
2-2.3.1 Glycerol cell stock preparation
In previous test, the cell culture with good expression efficiency was prepared as glycerol stock for stable yield and convenient storage. When OD600 of the cell culture exceeded 0.6, 600 μL of 50% sterilized glycerol was added to 600 μL of culture, resulting a 1.2 mL glycerol stock. The glycerol stock was kept at -30°C overnight and moved to -80°C refrigerator for long term storage.
2-2.3.2 Expression of recombinant ubiquitin protein
100 μL of glycerol stock was added to 100 mL TB medium (17.6 mM Na2HPO4,
7.4 mM KH2PO4, 137 mM NaCl, 2% peptone, 1.5% yeast extract, pH 7.4) with 10 mM glucose and 100 μg/mL ampicillin, and incubated overnight at 37°C, 250 rpm. The
culture was inoculated equally to four 2-liter flasks with 500 ml TB medium (also with 10 mM glucose and 100 μg/mL ampicillin), and the resulting 2L culture was further
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incubated at the same condition for 3 hours. Protein expression was induced by 1 mM IPTG for 4 hours at 37°C, 250 rpm. After induction, the culture was centrifuged (8,950 g for 30 min) and the cell pellet was collected. Then we resuspended the cell pellet with lysis buffer (40 mM Tris, 150 mM NaCl, pH 8.0). Cell lysis was done by adding 0.4 X CellLytic B (Sigma-Aldrich, USA), 150 μg/mL lysozyme, 10 μg/mL DNaseI, 5 mM MgCl2 and 1 mM PMSF and stirring for 30 minutes at room temperature. Then the cell lysate was centrifuged at 30,000 g for 30 minutes at 4°C and the supernatant was collected for further purification.
2-2.3.3 Ammonium sulfate precipitation
Next step, ammonium sulfate precipitation was applied to give a preliminary separation of different proteins in the supernatant collected before. For every 100 mL of cell lysate supernatant, 24.3 g of ammonium sulfate was added to give the solution of 40% ammonium sulfate saturation at 25°C. The solution was centrifuged at 10,000 g for 30 minutes at 4°C. Since ubiquitin remained soluble in this step, we collected the supernatant and keep adding ammonium sulfate. This time we added 28.5 g of ammonium sulfate for every 100 mL of supernatant, to give the solution of 80%
saturation. Ubiquitin would be precipitated at this step, therefore the solution was centrifuged again at 10,000 g for 30 minutes at 4°C, and the pellet was collected and
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dialysis (Cellu-Sep T1 3500 MWCO dialysis membrane; Membrane Filtration Products, USA) overnight in IEC A buffer (50 mM acetic acid, 50 mM sodium acetate, pH 4.7).
Some proteins became insoluble in this step and appeared as precipitant (whereas ubiquitin could be dissolved in the buffer), so the sample was centrifuged at 30,000 g for 30 minutes at 4°C, and the supernatant was collected.
2-2.3.4 Ion exchange chromatography and size exclusion chromatography
In ion exchange chromatography (IEC) and size exclusion chromatography (SEC), Ä KTA Protein Purification Systems (GE Healthcare, UK) were applied. We
concentrated the supernatant from last step to less than 50 mL and then filtered it by 0.22 μm filter (33mm; EMD Millipore, USA) for IEC. HiPrep SP Sepharose XL 16/10
cation-exchange column (GE Healthcare, UK) was used to purify mutated ubiquitin.
The buffer gradient was from 100% A buffer (50 mM acetic acid, 50 mM sodium acetate, pH 4.7) to 100% B buffer (50 mM acetic acid, 50 mM sodium acetate, 1M sodium chloride pH 4.7). Ubiquitin bound to negative-charged cation exchange columns and eluted at around 30% to 40% B buffer. Then we concentrated the fractions with ubiquitin to less than 5 mL and performed SEC. 1.6/60 cm Superdex-75 column (GE Healthcare, UK) and SEC buffer (150 mM sodium phosphate buffer, 1 mM DTT, pH 7.0) were used to remove the contaminations in the IEC flow through. 2 column volume
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(CV) of SEC buffer was pumped into the column to elute ubiquitin. The protein was eluted between 1.4 and 1.7 CV. Then the eluate was collected and dialyzed in 0.1% TFA.
Finally, the sample was lyophilized and the resulting ubiquitin powder was kept at -30°C.
2-2.4 Fluorescence dye labeling and purification
One vial of Alexa Fluor 488® C5 maleimide dye (1 mg; Life Technologies, USA) was dissolved in 100 μL DMSO to make a 13.9 mM stock. Ubiquitin powder prepared
previouly was dissolved in 30 mM sodium phosphate buffer (pH 7.0) to form a 30 μM ubiquitin solution and then filtered by 0.22 μm filter (33mm; EMD Millipore, USA). 1
mL of filtered solution was taken and 1 μL of 100 mM TCEP was added to it. The sample was degassed, then 5 μL of Alexa-488 stock was added and mixed gently. The
conjugation reaction was kept away from light at 25°C for 1.5 hours. Since Alexa-488 free dye eluted at approximately the same time as ubiquitin, dialysis was performed to remove free dye. About 1 mL of dye-labeled ubiquitin sample was kept away from light and dialysis in 500 mL of distilled water overnight by using Cellu-Sep T1 3500 MWCO dialysis membrane (Membrane Filtration Products, USA).
Next, reversed-phase HPLC was applied to separate the products. It was performed
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by using C18 column (DiscoveryBIO Wide Pore C18, 25 cm × 10 µm, Supelco, Sigma-Aldrich, USA) with Agilent HPLC system, and buffer A (94.9% water, 5%
acetonitrile, 0.1% TFA) and buffer B (99.9% acetonitrile, 0.1% TFA) were used. The purification condition was set with a gradient from 25% to 55% of B buffer within 30 minutes. Dye-labeled ubiquitin would be eluted at about 30% B buffer and could be separated from unlabeled protein. Purified products were collected and lyophilized, then stored at -30°C. The identification of dye-labeled ubiquitin was done with ESI-TOF mass spectrometry by Waters LCT Premier XE time-of-flight benchtop mass spectrometer (Waters, UK) in Mass Spectrometry Service Center, Academia Sinica.
2-2.5 Enzyme expression and purification
2-2.5.1 Expression of yeast E2 and E3 protein
Plasmid constructs of yeast E2 ubiquitin conjugating enzyme, Ubc5, and E3 ubiquitin ligase, Rsp5, were kindly provided by Dr. Hung-Ta Chen, Institute of Molecular Biology, Academia Sinica. Both these two constructs has an N-terminal hexahistidine tag following a SUMO-1 tag for purification purposes (figure 2.1).
Plasmids were transformed into E. coli. BL21 StarTM (DE3) to make glycerol stock (The methods are the same as 2-2.2 and 2-2.3.1). For each enzyme (both E2 and E3), 100 μL
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of glycerol stock was inoculated into 100 mL of sterile LB/ampicillin medium, and the culture was incubated overnight at 37°C, 250 rpm. On the next day, 20 mL of the overnight culture was subcultured into 500 mL sterile LB/ampicillin medium and incubated at the same conditions. After the absorbance OD600 of the culture exceeded 0.6 (about 3 hours later), 500 μL of 1M IPTG was added to give a final concentration of 1 mM for induced expression. 4 hours later, the culture was centrifuged (8,950 g for 30 min) and the supernatant was discarded. The cell pellet was kept in -80°C refrigerator until the next step was performed.
Figure 2.1 Constructs of yeast Ubc5 and Rsp5
Hexahistidine (6 His) tag is used for IMAC purification (described in chapter 2-2.5.2), and SUMO tag can undergo SUMO cleavage (chapter 2-2.5.3) to get pure products.
2-2.5.2 Immobilized metal-ion affinity chromatography (IMAC)
The cell pellet was resuspended with 30 mL of lysis buffer for IMAC (20 mM
HEPES, 500 mM NaCl, 10 μM ZnCl2, 2.5 mM imidazole, 0.5 mM TCEP, 2 mM MgCl2, 0.2 mM PMSF, 0.3 mg/mL lysozyme, 2 μg/mL2 DNaseI, pH 8.0) and then lysed with
three cycles of freezing and thawing (freezing with liquid nitrogen and thawing with 37°C water bath). Cell lysate was centrifuged at 30,000 g for 30 minutes at 4°C, and the
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supernatant was collected. Pre-packed column with Ni2+-charged Chelating Sepharose
Fast Flow (GE healthcare, UK) was used and balanced with IMAC A buffer (20 mM HEPES, 500 mM NaCl, 10 μM ZnCl2, 2.5 mM imidazole , 0.5 mM TCEP, pH 8.0)
beforehand. Filtered supernatant was applied to the column and the column was shaken for 30 minutes at 4°C to enhance the specific binding. The flow through was discarded, and 5 CV of IMAC D buffer (20 mM HEPES, 500 mM NaCl, 10 μM ZnCl2, 30 mM imidazole , 0.5 mM TCEP, pH 8.0) was applied twice to wash out nonspecific, loosely
binding proteins. Finally, hexahistidine-tagged proteins were eluted by 5 CV of IMAC E buffer (20 mM HEPES, 500 mM NaCl, 10 μM ZnCl2, 500 mM imidazole , 0.5 mM
TCEP, pH 8.0).
2-2.5.3 SUMO-tag cleavage and enzyme purification
Two samples, IMAC eluate with Ubc5 and eluate with Rsp5 from last step were dialyzed in IMAC A buffer to reduce imidazole concentration. Then the samples were
concentrated 10 folds or more. To remove N-terminal hexahistidine tag and SUMO tag, 150 μL of concentrated sample was added with 290 μL of distilled water, 50 μL of 10×
SUMO protease buffer (500 mM Tris-HCl, pH 8.0, 2% Igepal (NP-40), 10 mM DTT) and 10 μL SUMO protease Ulp1 (1U/μL; Invitrogen, Life Technologies, USA). The
final buffer condition was 150 mM NaCl (from IMAC A buffer), 50 mM Tris-HCl, 0.2%
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Igepal (NP-40), 1 mM DTT, pH 8.0, and the reaction was held at 4°C overnight.
After overnight reaction, a second round of IMAC was performed to separate the
cleavage product and other proteins in the sample. The 500 μL reacted sample was mixed with 15 mL of IMAC B buffer (20 mM HEPES, 500 mM NaCl, 10 μM ZnCl2, 10
mM imidazole , 0.5 mM TCEP, pH 8.0) and applied to B buffer pre-balanced Ni2+-charged column. After shaken for 30 minutes at 4°C, the flow through was
collected, and the sample was then eluted by IMAC C buffer (20 mM HEPES, 500 mM NaCl, 10 μM ZnCl2, 20 mM imidazole , 0.5 mM TCEP, pH 8.0) and IMAC D buffer
(the same as D buffer in 2-2.5.2) sequentially. In E2 purification, Ubc5 didn’t bind to the column and would appear in flow through; whereas in E3 purification, most Rsp5 would be eluted by IMAC C buffer. The collected flow through and eluate with high purity of E2 or E3 were dialyzed in 1× ubiquitination buffer (50 mM Tris-HCl, 10 mM MgCl2, 50 μM ZnCl2, 1 mM DTT, pH 7.5), concentrated, and then store at -30°C until use.
2-2.6 Ubiquitination assay with dye-labeled ubiquitin
When dye-labeled ubiquitin mutants and E2, E3 enzymes were ready to use, we performed in vitro ubiquitination. Our substrate was yeast RNA polymerase II (Pol II),
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which was kindly provided by Dr. Wei-Hau Chang, Institute of Chemistry, Academia
Sinica. 300 ng Pol II, 0.5 μg human E1 activating enzyme UBE1 (Sigma-Aldrich, USA), 1 μg yeast E2 Ubc5, 300 ng yeast E3 Rsp5, 2 μg ubiquitin (wild-type or dye-labeled)
were mixed in ubiquitination buffer (50 mM Tris, 10 mM MgCl2, 50 μm ZnCl2, 1 mM DTT, pH 7.5) and 2 mM ATP, with a total volume of 30 μL. Ubiquitin would conjugate to Pol II subunit Rpb1 in this reaction. After 30°C incubation with mild shaking for overnight, silver staining, Western blotting and fluorescent imaging were used to investigate the efficacy of ubiquitination.
2-2.7 Gel electrophoresis and imaging
20 μL of each sample was mixed with 5 μL of 5× SDS loading dye (250 mM
Tris-HCl, 10% SDS, 50% glycerol, 10 mM EDTA, 0.25 mg/mL bromophenol blue, 25%
BME in distilled water), heated on dry bath at 95°C for 10 minutes. Then 10 μL of the sample was loaded into a well in gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 8% Bis-Tris polyacrylamide gel 1.0 mm (The protocol of this gel is listed below) under 150V for 35 minutes, or 4 – 12% gradient gel (NuPAGE® Novex 4-12% Bis-Tris Gel 1.0 mm, 10 Well; Life Technologies, USA) under 200V for 35 minutes.
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2-2.7.1 Fluorescence imaging
When electrophoresis was done, the proteins on gel were fixed by fixing buffer (30% ethanol, 10% acetic acid in distilled water) for 30 minutes. The gel was then rinsed twice in 20% ethanol, for 10 minutes for each wash, and then twice in water, for 10 minutes for each wash. After that, the gel was scanned by fluorescent scanner Amersham TyphoonTM 9200 Imager (GE Healthcare, UK). The laser and emission filter were set to 488 nm and 520 nm, respectively. The sensitivity was set to normal, and the pixel size was set to 100 microns or 200 microns.
2-2.7.2 Silver stain
After fluorescence imagine, the gel could be used directly to do silver stain. The gel was sensitized by 0.8 mM sodium thiosulfate for 1 minute and then rinsed twice in water for 1 minute for each wash. Next, the gel was soaked in 0.2% silver nitrate (12
mM) for 20 minutes. After silver impregnation, the gel was dipped in water for 10 seconds and then moved to developer (3% sodium carbonate, 250 μl formalin and 125 μl 10% sodium thiosulfate per liter) for 5 minutes. Last step, the gel was immersed in
stop solution (4% Tris and 2% acetic acid in water). The detailed protocol of silver stain was published by Chevallet et al. [36].
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2-2.7.3 Western blot
Another gel just finished electrophoresis was used to do Western blot. First, the
proteins on gel were transferred to Whatman® Protran® nitrocellulose membrane BA85, pore size 0.45 μm (PerkinElmer, USA), in Western blot transfer buffer (5.8 g glycine,
2.9 g Tris, 10% methanol per liter) under 300 mA for 80 minutes. Meanwhile, 1 liter of wash buffer (8.7 g sodium chloride, 6 g Tris, 1 mL Tween-20 in 1 liter buffer, pH 7.5) was also prepared. Later, 10 mL of blocking buffer (wash buffer plus 5% milk powder) was used to avoid nonspecific bindings on the membrane. After 30 minutes of blocking, 1st antibody (rabbit anti-ubiquitin antibody; Sigma-Aldrich, USA) was added directly, and binding overnight at 4°C with shaking. On the next day, the membrane was washed
3 times in wash buffer, 5 minutes each time. Then 10 mL of blocking buffer was added, in addition with 2 μL of 2nd antibody (goat anti-rabbit-IgG antibody, HRP peroxidase
conjugated; EMD Millipore, USA), binding for 1 hour with shaking. After additional 3 times of washing (5 minutes each), Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, USA) was used for chemiluminescent detection. Then FUJI SuperRX developing film (Fujifilm, Japan) was used, and the developing time was set from 20 seceonds to 3 minutes. KODAK RP X-OMAT Developer and Replenisher (Eastman Kodak, USA) was used to develop the film.
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