Expression and purification of double Strep-tagged NupC
Vincent Postis
Materials:

Bicinchoninic acid (BCA) reagent (Pierce, 23223)

Escherichia coli strain C43 (DE3) harbouring the plasmid pRARE2 (Novagen) (home
made)

Tris base (FW 121.14) (Melford, B2005)

Carbenicillin (Melford, C0109)

Glucose (Ducheta Biochemie, G0802.1000)

Agar (Oxoid, LP0011)

Glycerol (Melford, G1345)

d-Desthiobiotin (FW 214.26) (Sigma, D1411)

HABA (2-(p-Hydroxyphenylazo)benzoic acid, FW 242.23) (Alfa Aesar, A10333)

IPTG (isopropyl-β-D-thiogalactoside, FW 238.3) (Generon, C9H1805S)

Protease inhibitor cocktail without EDTA (Roche, 04693159001)

NaCl (FW 58.44) (Fisher, S3160/63)

n-dodecyl-β-D-maltoside (DDM), (Melford, B2010)

Strep-Tactin® Superflow® 50% suspension (IBA 2-1206-002)

Tryptone (Melford, T1332)

Vivaspin MWCO 100000 (Sartorius, VS0241)

Yeast extract (Melford, Y1333)
Media and solutions:

Lysogeny Broth (LB)-medium. Per litre:
Dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in H2O to give a final volume
of 1 litre then sterilise by autoclaving.

LB-agar: LB supplemented with 1.5 % agar.

10X Phosphate-buffered saline pH 7.4 (10X PBS; 100 mM Na2HPO4, 18 mM KH2PO4,
1370 mM NaCl, 40 mM KCl, pH 7.4). Per litre:
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-2Dissolve 80 g NaCl, 3 g KCl, 14.4 g Na2HPO4 and 2.4 g KH2PO4 in 800 mL water,
adjust pH to 7.4 and make up to the volume to 1 litre. Sterilize by autoclaving.

1 M Tris-HCl, pH 7.0
Dissolve 60.57 g Tris in 400 mL H2O. Adjust the pH to 7.0 with HCl then use a
volumetric flask to make up to a final volume of 500 mL.

0.5 M EDTA, pH 7.0
Dissolve 9.307g Na2EDTA.2H2O (FW 372.25) in about 40 mL H2O. Warming will
probably be necessary. Cool to room temperature, adjust pH to 7.0 by addition of 1 M
NaOH, then make up volume to 50 mL. Store at 4 °C, or frozen for extended periods.

IPTG (1 M):
Dissolve 1.906 g IPTG to a final volume of 8 mL in milli-Q H2O then sterilise by
passage through a 0.22 µm filter. Dispense into 1 mL aliquots and store at -20°C.

3 M NaCl
Dissolve 87.66 g NaCl in ~400 mL H2O then make up the volume to 500 mL.

50% w/v glycerol
Weigh 100 g glycerol into a 200 mL volumetric flask then make up to a final volume of
200 mL.

200 mM HABA
Weigh 2.422 g HABA into a 50 mL volumetric flask. Dissolve in H2O then make up to a
final volume of 50 mL and store at 4°C.

2 x Solubilisation buffer (100 mM Tris-HCl, pH 7.0, containing 2 mM EDTA, 200 mM
NaCl and Complete Protease Inhibitor Cocktail). Per 40 mL:
Mix 2.66 mL 3 M NaCl, 4 mL 1 M Tris-HCl, pH 7.0, 160 μL 0.5 M EDTA, pH 7.0 and 1
tablet protease inhibitor cocktail then dissolve and dilute to give a final volume of 40 mL
in H2O.
Note - this buffer lacks detergent, which is added separately.

Wash buffer (50 mM Tris-HCl, pH 7.0, containing 1 mM EDTA, 100 mM NaCl, 5%
glycerol, 0.05% DDM). Per 10 mL:
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-3Mix 0.333 mL 3 M NaCl, 0.5 mL 1 M Tris-HCl, pH 7.0, 20 μL 0.5 M EDTA and 20 µL
25% DDM then dilute to give a final volume of 10 mL in H2O.
Note DDM is very expensive so only make up the required amounts of this and other
buffers.

Binding dilution buffer (50 mM Tris-HCl, pH 7 .0, containing 1 mM EDTA, 100 mM NaCl
and 10% glycerol)
Mix 2.66 mL 3 M NaCl, 4 mL 1 M Tris-HCl, pH 7.0, 160 μL 0.5 M EDTA and 16 mL
50% glycerol then dilute to give a final volume of 80 mL in H2O.

Elution buffer (50 mM Tris-HCl, pH 7.0, containing 1 mM EDTA, 100 mM NaCl, 2.5 mM
d-Desthiobiotin, 5% glycerol and 0.05% DDM). Per 5 mL:
Dilute 0.166 mL 3 M NaCl, 0.25 mL 1 M Tris-HCl, pH 7.0, 10 μl 0.5 M EDTA, 2.7 mg dDesthiobiotin and 10 µL 25% DDM to give a final volume of 5 mL in H2O.

Regeneration buffer (50 mM Tris-HCl, pH 7.0, containing 1 mM EDTA, 100 mM NaCl
and 1mM HABA). Per 100 mL:
Dilute 3.333 mL 3 M NaCl, 5 mL 1 M Tris-HCl, pH 7.0, 200 μL 0.5 M EDTA and 0.5 mL
200 mM HABA to give a final volume of 100 mL in H2O.
Expression and isolation of membranes
Day 1: Transform E. coli strain C43(DE3) harbouring the plasmid pRARE2 (Novagen), with
pBPT0217-CTS2. Turn the PCR machine on and start the program ‘TRANSFM’ ([4C-∞;
4C-30 min; 42C-30 sec; 4C-2 min; 4C-∞; 37C-1 h; 4C-∞]). Thaw 50 μL competent cells in
a 0.5 mL Eppendorf tube on ice then add 1 μL plasmid DNA (ideally containing ≤ 50 ng
DNA). Transfer the tube to the PCR machine and click on ‘SKIP’. When the machine has
stopped at the second 4C-∞; step, add 250 µL of sterile pre-warmed LB medium lacking
antibiotics and click on ‘SKIP’. After the end of the 37°C incubation, spread all the
suspension on an LB-agar plate supplemented with carbenicillin (100 µg/mL) and incubate
overnight at 37°C.
Note, it is not necessary to select for the presence of pRARE2 for EcNupC i.e. it is not
necessary to have chloramphenicol in the plates.
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-4Day 2: inoculate 50 mL LB medium, supplemented with carbenicillin (100 µg/mL) and 1%
glucose, with a single colony from the plate and incubate overnight at 37°C with orbital
shaking at 200 rpm in a 250 mL flask.
Day 3: Measure the D600nm of the overnight culture and inoculate 2.5 L baffled flasks, each
containing 500 mL LB medium supplemented with carbenicillin (100 µg/mL), to give a
theoretical D600nm value of 0.05 and incubate at 37°C with orbital shaking at 200 rpm.
Monitor the D600nm and when it reaches 0.7 induce protein expression by adding IPTG to
give a final concentration of 0.5 mM. Incubate for a further 3 h under the same conditions.
Harvest the cells using a Beckman Coulter Avanti J-26 XP centrifuge (9000 g 40 min at 4
°C, using JLA8.1000 rotor). Resuspend the cell pellets (6 ml buffer/g cells) in ice-cold PBS
(1X), pH 7.4, containing 1 cOmplete, Mini Protease Inhibitor Cocktail Tablet per 50 ml.
Homogenise the cells using a T18 basic ULTRA-TURRAX® homogeniser and then disrupt
using a TS series continuous cell disruptor (Constant Systems, UK) operating at 30 kPSI
and 4°C. Pellet the cell debris at 14000 g for 45 min in a Beckman Coulter Avanti J-26 XP
(JLA16.250) centrifuge, followed by ultracentrifugation at 100000 g (Ti45 rotor, Beckmann)
for 2 h at 4 °C to collect the membranes. Resuspend the latter in a minimal volume (a few
mL) of PBS (1X), pH 7.4 using a glass/Teflon homogeniser. Take a small sample for
measurement of the protein concentration using the BCA assay, then snap-freeze in
droplets in liquid nitrogen (take appropriate precautions - see COSHH form!!) and store at 80°C.
Typical membrane protein concentration will be between 30 and 35 mg/mL
Purification
All purification steps should be done at 4°C
Dilute 140 mg of membrane protein with 35 mL ice-cold 2 x solubilisation buffer then add
ice-cold milli-Q water to give a final volume of 67.2 mL. Note, if the membrane suspension
is lumpy, homogenise using a glass/Teflon homogeniser until homogeneous. Then add 2.8
mL 25% DDM, to give a final detergent concentration of 1% and a protein concentration of
2 mg/mL; incubate on a roller mixer for 1 h. Take a small sample (e.g. 100 μL) and snapfreeze for subsequent SDS-PAGE/western blotting to assess the “total” amount of EcNupC
in the starting material.
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-5Note, store snap-frozen samples at -80 °C rather than at -20°C to prevent protein
degradation.
Pellet the insoluble fraction in an ultracentrifuge (Ti45 rotor, Beckmann) at 100000 g for 1
h. Carefully remove the supernatant from the pellet, measure its volume and keep this on
ice. If desired, solubilise the pellet in gel sample buffer (it is hard to get an even
resuspension if ordinary buffer is used) and snap-freeze for subsequent SDSPAGE/western blotting to assess the amount of EcNupC that has not been solubilised.
Similarly, take a small sample (e.g. 100 μL) of the supernatant to assess the amount of
EcNupC that has been solubilised.
Note, if you have not used the ultracentrifuge before you MUST be trained first by an
experienced user.
Pre-equilibrate 1 mL Strep-Tactin® Superflow® resin slurry (50% slurry i.e. 0.5 mL packed
resin) in a 50 mL Falcon tube by washing three times with 10 mL milli-Q water and then
three times with 3 mL wash buffer. Do this by gently inverting the tube then centrifuging at
700 g for 5 min each time in an Eppendorf 5804 R centrifuge. Then mix the supernatant
from the ultracentrifugation step with an equal volume of Binding dilution buffer before
adding to the resin and allowing protein binding to occur overnight with gentle mixing on a
roller mixer.
Nominal capacity of the resin is 50 to 100 nmol per mL packed volume, so 0.5 mL resin
should bind up to 50 nmol EcNupC i.e. up to about 2.4 mg
The next day centrifuge again at 700 g for 5 min (in an Eppendorf 5804 R centrifuge),
remove and discard the supernatant (but first keep a sample for SDS-PAGE/western
blotting to assess the amount of EcNupC that has remained unbound).
Next pack the resin into a column of appropriate dimensions (e.g. in this case Disposable
Plastic Columns, Thermo-Pierce Cat. 29920). Then wash the resin under gravity with 5 mL
(i.e. 10 column volumes (CV)) of wash buffer. Close the column outlet and gently add 0.3
mL elution buffer, without disturbing the top of the resin. Open the column again and
collect 0.3 mL of the eluate (measure using the 0.5 mL mark on the Eppendorf tube),
before closing the column outlet again. This fraction should correspond to the wash buffer
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-6initially present on the column i.e. it should not contain eluted protein, but it should be kept
just in case!
Next perform the following steps:
(a) With the column outlet still closed, add 0.5 mL elution buffer, re-suspend the resin with
a pipette tip and incubate for 10 min at 4 ºC on rotary mixer.
(b) Place the column in the vertical position again, open the outlet and collect the eluate
plus that generated by subsequent gentle addition of 0.5 mL elution buffer, in a single
tube. Close the outlet again. Measure the A280nm of the eluted fraction to provide an
indication of protein concentration, using elution buffer as the blank.
Repeat steps (a) and (b) until all the protein has eluted. Pool the peak fractions, as
indicated by their absorbance.
Then, to remove the d-Desthiobiotin, dialyse overnight in the cold room against 50 mM
Tris-HCl, pH 7.0, containing 1mM EDTA, 100 mM NaCl, 5% glycerol and 0.05% DDM.
Note, calculate the theoretical final concentration of d-Desthiobiotin, which will depend
upon the volume of sample dialysed, and make sure this is sufficiently low not to interfere
with subsequent experiments. Add a further dialysis step if necessary.
If necessary, concentrate the dialysed sample using a Vivaspin concentrator with a 100
kDa molecular weight cut-off by centrifugation at 3800 g at 4 °C.
2. Resin regeneration
For regeneration, wash the column 3 times with 5 CV of Regeneration buffer (3 times
2.5 mL in this case). The colour change from yellow to red indicates the regeneration
process and the intensity of the red colour is an indicator of the column activity status.
Regeneration is complete when the red colour on the bottom of the column has the
same intensity as on top of the column. If this is not the case use more Regeneration
buffer.
Store the column at 4 °C overlaid with 1 mL of Regeneration buffer.
Remove the Regeneration buffer by washing the column 2 times with 4 CV of Wash
buffer (2 times 2 mL in this case) before the next purification run.
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-7This protocol is for expression and purification of NupC from Escherichia coli (EcNupC),
bearing a C-terminal double Strep-tag II sequence, encoded by the plasmid pBPT0217CTS2. The sequence of the tagged protein is as follows:
MASDRVLHFVLALAVVAILALLVSSDRKKIRIRYVIQLLVIEVLLAWFFLNSDVGLGFVKGFS
EMFEKLLGFANEGTNFVFGSMNDQGLAFFFLKVLCPIVFISALIGILQHIRVLPVIIRAIGFLL
SKVNGMGKLESFNAVSSLILGQSENFIAYKDILGKISRNRMYTMAATAMSTVSMSIVGAYM
TMLEPKYVVAALVLNMFSTFIVLSLINPYRVDASEENIQMSNLHEGQSFFEMLGEYILAGFK
VAIIVAAMLIGFIALIAALNALFATVTGWFGYSISFQGILGYIFYPIAWVMGVPSSEALQVGSI
MATKLVSNEFVAMMDLQKIASTLSPRAEGIISVFLVSFANFSSIGIIAGAVKGLNEEQGNVV
SRFGLKLVYGSTLVSVLSASIAALVLPAGSAGENLYFQGSGGGGAGSAWSHPQFEKGG
GSGGGSGGGSWSHPQFEK
…… Strep tag II sequence
…… Linker between strep tags
…… Linker used in IBA vectors
…… Sequences encoded by NheI and SbfI restriction sites respectively
…… TEV recognition site
 TEV Cleavage site
The wild-type NupC sequence is shown in italics
Length
Molecular Weight
1 microgram
Molar Extinction
coefficient
1 A280nm unit
A280nm of 1 mg/ml
solution
Isoelectric Point
Charge at pH 7
453 aa
48490.84
20.622
pmol
43930
1.10 mg/ml
0.91 AU
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7.94
1.22