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
}){kind=link}
}){kind=link}
