Biomod/2011/Slovenia/BioNanoWizards/methprotprodandisol

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Protein production and isolation
 

Transformation of BL21(DE3)pLysS


All our proteins were produced by transformed Escherichia coli BL21(DE3)pLysS. Transformation was performed by slowly thawing aliquots of competent bacterial cells on ice for 30 min, adding 1 μl of plasmid DNA to each microcentrifuge tube and leaving the mixtures on ice for another 30 min. Heat shock was performed next by heating the cells to 42 °C for 3 to 5 min. Immediately after that they were cooled on ice for 1 to 2 minutes. 1 ml of LB medium was then added to each microcentrifuge tube. The cultures were incubated for 1 h at 37 °C while shaking and then centrifuged for 3 min at 7000 rpm on a tabletop microcentrifuge. Most of the supernatant medium was removed and the rest was used to resuspend the pelleted cells, which were then plated on LB-agar plates with kanamycin and incubated overnight at 37 °C. A successful transformation resulted in many colonies of transformed protein producing cells.


Protein expression

A single colony was picked with a sterile toothpick, which was used to inoculate 100 to 125 ml of growth medium in a flask. Alternatively, a glycerol stock of the bacteria in question could be used. Inoculated medium was shaken overnight at 37 °C and 180 rpm and its OD600 was measured in the morning. A part of the overnight culture was used as an inoculum for larger flasks so that the starting OD600 of larger volumes of production medium was 0.10-0.15. Flasks were shaked at 37 °C and 160 or 180 rpm. When OD600 reached 0.7-0.9, production of proteins was induced by adding IPTG to a final concentration of 1 mM. After several hours of shaking at 37 °C and 160/180 rpm biomass was harvested by centrifugation for 10 min at 5500 rpm in a BeckmanCoulter centrifuge with JA-10 rotor. Pelleted biomass was either frozen or directly processed further.

GST-ZFP fusions were overexpressed in bacteria grown in LB medium supplemented with kanamycin (100mg/L), ZnCl2(0.5 mM) and induced with 1 mM IPTG, while 2xYT growth medium supplemented with 10g/L glucose, kanamycin (100mg/L), ZnCl2(0.5 mM) and induced with 1 mM IPTG was used for overexpression of MBP-ZFP fusions as well as twin ZFP protein tethers and BRET fusions, namely MBP-RLuc-2C7 and MBP-YFP-AZPA4.
 


Cell lysis

After pelleting bacterial cells were lyzed by resuspending pelleted biomass in lysis buffer (20 mM Tris-HCl, 100 mM NaCl, 0.1 % deoxycholic acid, 100 μM ZnCl2, 1:500 CPI-His tag and 1 mM DTT at pH 7.5 - used for GST-ZFP chimeras - or 25mM HEPES, 300mM NaCl, 0.2% Triton X-100, 1mM TCEP-HCl, 500μM ZnCl2, 1:500 CPI-His tag at pH 8.0 for MBP-ZFP fusions as well as twin ZFP protein tethers and BRET fusions, namely MBP-RLuc-2C7 and MBP-YFP-AZPA4). After 20 min the suspensions were sonicated for approximately 10 min with a pulse every second or third second until they became clear lysates. Lysates were then centrifuged for 20 min at 4 °C and 12000 rpm in a Hettich Universal 320 R centrifuge. Supernatants were separated from pellets of cellular debris and inclusion bodies. Samples for SDS-PAGE and Western blot were taken from both fractions and the rest frozen.
 


SDS page

SDS-PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. In this method protein samples are exposed to sodium dodecyl sulfate, a negatively charged detergent that denatures proteins and gives them a uniformly negative charge resulting in a defined charge/mass ratio. Samples are loaded onto a polyacrylamide gel and exposed to an electric field. Negatively charged protein-SDS complexes travel through the gel towards a positive electrode. The samples concentrate in stacking gel and start separating when they enter separating gel according to their size because the bigger the molecules are, the harder they move through gel. The gel can then be stained to visualize proteins with Coomassie Brilliant Blue (CBB) or used in Western blotting.

Polyacrylamide minigels were made with BioRad equipment for use with MiniProtean II vertical electrophoresis system. In our experiments stacking gels with 4 % and separating gels with 10 - 15 % acrylamide were used. Separating gels were buffered with 375 mM Tris-HCl at pH 8.8 and stacking gels with 125 mM Tris-HCl at pH 6.8. They both contained 0.1 % SDS and were polymerized using 0.05 % (w/V) APS and 0.05 % (separating) or 0.1 % (stacking) TEMED. The separating gel mixture was poured between two glass plates usually 1 mm apart and covered with 200 ul isopropanol. After at least 30 minutes the mixture for stacking gel was prepared, isopropanol removed with filter papers, stacking gel mixture poured and a comb for sample pockets inserted between the glass plates. After at least 30 minutes the gel was ready to be used or wrapped into a moist paper towel and aluminum foil and refrigerated for later use.

Samples were prepared by adding a 4x loading buffer that contains 4 % (w/V) SDS, 125 mM Tris-HCl at pH 6.8, 40 % (vol.) glycerol, 10 % (w/V) beta-mercaptoethanol and 0.1 % (trace amounts) of bromophenol blue. Samples with 1x loading buffer concentration were loaded into pockets of the stacking gel. Electrophoresis was run in 25 mM Tris, 192 mM glycine and 0.1 % (w/V) SDS at pH 8.3 for approximately 50 minutes at 200 V. Afterwards the gel was stained with CBB and destained with a mixture of 30 % ethanol and 20 % acetic acid until protein bands were clearly visible.


Western blot

Western blotting is a general term for describing methods of transferring proteins from an acrylamide gel to a nitrocellulose or PVDF membrane for probing with antibodies.

We used two types of Western blotting - "wet blotting" and iBlot(R). The first consists of placing a prerun gel in a sandwich of soaked sponges and filter papers with a nitrocellulose HybondTM ECL being on the anode side of the prerun gel. BioRad PowerPacTM 3000 setup was used for this type of blotting. Process was performed in cold transfer buffer consisting of 25 mM Tris-HCl, 192 mM glycine and 20 % methanol at 350 mA for 50 minutes. The second type is actually a variant of electroblotting and was performed according to manufacturer's instructions. Using the second type, proteins are transferred to the membrane in 7 minutes.

After blotting the membrane was incubated in 5 % skimmed milk in 1x TBS buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) for 1.5 h at room temperature. This step prevents non-specific binding of antibodies to the membrane in later steps thus preventing high background signal or false positives. Next the membrane was incubated in a plastic bag with 6 ml mouse anti-(H)4 IgG antibodies (Qiagen, Tetra-His antibody), diluted 1:2000 with 1 % skimmed milk in 1x TBS, for 1 h at room temperature while shaking. Excess antibodies were washed away in 4 steps of 5 minute shaking in 1x TBS buffer. Membrane was then incubated in a plastic bag with 6 ml secondary goat anti-mouse IgG-HRP antibodies (Santa Cruz Biotechnology), diluted 1:3000 with 1 % skimmed milk in 1x TBS, for 50 minutes at room temperature while shaking. Excess antibodies were again washed away in 4 steps of 5 minute shaking in 1x TBS buffer. The membrane was then soaked in ECL reagent for 1 minute and photographed in Syngene G:Box gel imaging system. Horseradish peroxidase catalyzed reaction produced light at His-tagged protein location.
 


Protein isolation

After production of proteins and their presence in lysate supernatants had been confirmed by Western blot, supernatants were thawed and centrifuged once more for 20 min at 4 °C and 12000 rpm. Cleared supernatants were then loaded onto 3 ml Qiagen Ni-NTA agarose columns, which were then shaken overnight at 4 °C. Before use, Ni-NTA agarose columns were washed with regeneration buffer containing 6 M guanidine hydrochloride, 100 mM Na3PO4, 10 mM Tris and 500 mM imidazole at pH 5.8. Columns were then washed with copious amounts of MQ water to remove denaturant and imidazole and in case of GST-ZFP fusions equilibrated with 10 column volumes of binding buffer consisting of 20 mM Tris-HCl, 50 mM NaCl, 10 μM ZnCl2 and 1 mM DTT (added just before use). In case of MBP-ZFP fusions, twin ZFP protein tethers and BRET fusions, namely MBP-RLuc-2C7 and MBP-YFP-AZPA4, the column was equilibrated with 10 volumes of binding buffer consisting of 25mM HEPES, 300mM NaCl, 0.2% Triton X-100, 1mM TCEP-HCl, 500μM ZnCl2, 1:500 CPI-His tag at pH 8.0. Supernatants were loaded on column and incubated with moderate shaking at 4°C overnight.

Next morning the supernatants were left to flow through the columns, which were then washed with binding buffer until A280 dropped to 0,02 or lower. Then, in case of GST-ZFP fusions, they were washed with a buffer of same composition as the binding buffer with addition of 20 mM imidazole. Columns were washed with this buffer until A280 dropped to 0,02 or lower. This step was repeated with buffers containing 50 mM imidazole and 100 mM imidazole. His-tagged proteins were eluted with a buffer containing 250 mM imidazole. Useful elution fractions collected in microcentrifuge tubes were joined together and dialyzed in 3500 MWCO dialysis membranes (Spectra/Por) against a buffer without imidazole, which in case of GST-ZFP fusions consisted of 20 mM Tris-HCl, 50 mM NaCl, 100 μM ZnCl2 and 1 mM DTT at pH 7.7.
MBP-ZFP fusions, twin ZFP protein tethers and BRET fusions were, after overnight shaking, treated the same as GST-ZFP fusions, except that buffer of a distinct composition was used for washing the column and later on elution of the proteins. The buffers' composition were as follows: 25 mM HEPES, 300 mM NaCl, 1mM DTT, 0.5 mM ZnCl2 with increasing concentrations of imidazole - 20, 50, 100 and 250 mM. MBP-ZFP fusions were dialyzed in 3500 MWCO dialysis membranes against MQ and twin ZFP protein tethers and BRET fusions with MBP against 10 mM Tris-HCl, 100 mM NaCl, 1mM DTT and 100 μM ZnCl2 at pH 8.0.

Attempts to isolate BRET fusions without MBP solubility tag were made under denaturing conditions (with 8 M urea being the denaturant) using buffers as follows. Binding buffer for column equilibration and the first washing step: 10 mM Tris, 100 mM, NaH2PO4, 8 M urea, pH 8.0. Subsequent wash buffers for gradual elution of both proteins were of the same composition as the binding buffer, except for the addition of imidazole in increasing concentration from 10, 50, 100 and 250 mM. The later had different pH as well - 5.8.

 

Proteins used in our project

Here is a summary of proteins characterized within our project. Estimated binding affinities for selected zinc finger proteins (ZFPs) are also presented and are based on the data from the literature.
 



ZFPs characteristics


Three finger ZFPs

ZFP name Number of amino acids Size [kDa] Isoelectric point (pI) Estimated Kd
sZif268 91 11.1 11.39 /
Zif268 87 10.3 9.81 2.0 nM (Papworth, 2003)
PBSII 86 9.8 9.48 /


Six finger ZFPs

ZFP name Number of amino acids Size [kDa] Isoelectric point (pI) Estimated Kd
2C7 180 21.4 9.98 0.46 nM (Liu, 1997)
6F6 177 20.8 10.18 10-20 pM (Papworth, 2003)
AZPA4 168 19.3 9.24 < 3pM (Sera, 2002)


Soluble ZFPs

Protein name Number of amino acids Size [kDa] Isoelectric point (pI)
GST-Zif268 324 37.8 8.41
GST-sZif268 324 38.4 9.31
GST-PBSII 323 37.3 8.22
GST-2C7 417 48.9 9.03
GST-6F6 414 48.3 9.13
GST-AZPA4 405 46.8 8.54
MBP-6F6 571 64.2 8.83
MBP-2C7 574 64.8 8.69


Twin ZFP protein tethers

Protein name Number of amino acids Size [kDa] Isoelectric point (pI)
2C7-MBP-6F6 756 86.03 9.23
AZPA4-MBP-6F6 744 83.93 8.99


BRET proteins

Protein name Number of amino acids Size [kDa] Isoelectric point (pI)
YFP-AZPA4 429 48.43 8.11
rLuc-2C7 513 59.52 7.37
MBP-YFP-AZPA4 813 90.43 6.58
MBP-rLuc-2C7 897 101.53 7.62




Liu Q et al (1997) Proc. Natl. Acad. Sci. USA 94: 5525-5530
Papworth M et al (2003) Proc. Natl. Acad. Sci. USA 100: 1621-1626
Sera T & Uranga C (2002) Biochemistry 41: 7074-7081
 

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