Expression of un-tagged NupC from Escherichia coli
Steve Baldwin, Vincent Postis, David Sharples
1. Materials:


















Bicinchoninic acid (BCA) reagent (Thermo, 23223)
CaCl2·2H2O (FW 147.02) (Fisher, BP510-100)
Carbenicillin (Melford, C0109)
Escherichia coli strain BL21(DE3)
Glucose (Sigma, G7528)
Agar (Oxoid, LP0011)
Glycerol (Melford, G1345)
IPTG (isopropyl-β-D-thiogalactoside (F.W. 238.3) (Generon, C9H1805S)
Protease inhibitor cocktail without EDTA (Roche, 04693159001)
MgSO4·7H2O (FW 246.47) (BDH, 101514Y)
NaCl (FW 58.44) (Fisher, S3160/63)
Na2HPO4 (FW 141.96) (Melford, S2002)
NH4Cl (FW 53.49) (Melford, A2042)
Na2EDTA.2H2O (FW 372.25) (Melford, E0511)
Sucrose (FW 342.3) (Melford, S0809)
Tris base (FW 121.14) (Melford, B2005)
Tryptone (Melford, T1332)
Yeast extract (Melford, Y1333)
Media and solutions:

100 mg/mL carbenicillin: Dissolve 500 mg carbenicillin in a final volume of 5 mL H2O,
filter sterilise through a 0.22 μm filter and store in the dark at -20°C.

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. Supplement with antibiotics and glucose as
required.

LB-agar: LB supplemented with 1.5 % agar.

40% (w/v) glucose:
Dissolve 40 g glucose in H2O to give a final volume of 100 mL. Filter sterilise through a
0.22 μm filter.

20X M9 salts:
Dissolve 120 g Na2HPO4, 60 g KH2PO4, 10 g NaCl and 20 g NH4Cl in 800 mL H2O and
adjust pH to 7.4 with 10 M NaOH. Make up volume to 1 litre then sterilise by
autoclaving.
-2
20% (w/v) casamino acids:
Dissolve 20 g casamino acids in H2O to give a final volume of 100 mL then sterilise by
autoclaving.

1 M CaCl2:
Dissolve 14.7 g CaCl2·2H2O in a final volume of 100 mL H2O then sterilise by
autoclaving or filtration through a 0.22 μm filter.

1 M MgSO4:
Dissolve 24.65 g MgSO4·7H2O in a final volume of 100 mL H2O then sterilise by
autoclaving or filtration through a 0.22 μm filter.

50% (w/v) glycerol:
Dissolve 100 g glycerol in H2O to give a final volume of 200 mL. Sterilise by
autoclaving.

M9 medium supplemented with 20 mM glycerol:
To 924 mL sterile H2O add 50 mL 20X M9 salts, 0.2 mL 1 M CaCl2, 2 mL 1 M MgSO4,
20 mL 20% casamino acids and 4 mL 50% glycerol. Add carbenicillin (100 μg/mL)
before use. Avoid CaCl2 precipitation by adding MgSO4 first.

IPTG (1 M):
Dissolve 1.906 g IPTG to a final volume of 8 mL in milli-Q H2O then filter sterilise
through a 0.22 µm filter. Dispense into 1 mL aliquots and store at -20°C.

0.5 M EDTA, pH 7.5
Dissolve 9.307g Na2EDTA.2H2O in about 40 mL H2O. Warming will probably be
necessary. Cool to room temperature, adjust pH to 7.5 by addition of 1 M NaOH, then
make up volume to 50 mL. Store at 4 °C, or frozen for extended periods.

20 mM Tris-HCl, 0.5 mM EDTA, pH 7.5 at 4°C. Per litre:
Dissolve 2.42 g Tris in 800 mL H2O, then adjust pH with 1M HCl to the following
values, depending upon the temperature of the buffer, so that it is 7.5 at 4°C (the
temperature coefficient of Tris is -0.028 pH/°C):
18°C
19°C
20°C
21°C
22°c
23°C
7.11
7.08
7.05
7.02
7.00
6.97
-324°C 6.94
25°C 6.91
Add 1 mL 0.5 M EDTA, pH 7.5 and then make up volume to 1 litre.

Tris-EDTA-Sucrose solutions for density gradient centrifugation:
Prepare these by dissolving sucrose in 20 mM Tris-HCl, 0.5 mM EDTA, pH 7.5 to
generate solutions with 55, 50, 45, 40, 35, 30 and 25 % (w/w) sucrose, e.g. for the
first, per 100 g, dissolve 55 g sucrose in 45 mL buffer. Store at -20°C. Defrost
overnight and gently mix by swirling before use.
Method:
Day 1:
Transform E. coli strain BL21(DE3) cells with pGJL16. 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 4Ce
pre-warmed LB medium lacking antibiotics and click on ‘SKIP’. Upon 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.
Day 2:
Inoculate 50 mL LB medium, supplemented with carbenicillin (100 µg/mL) and 1% glucose
(i.e. add 1.25 mL 40% glucose per 50 mL), 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:
1. Measure the D600nm of the overnight culture and inoculate 8x 2.5 L baffled flasks,
each containing 500 mL M9 medium supplemented with 20 mM glycerol and
carbenicillin (100 µg/mL), to give a theoretical D600nm value of 0.05 and incubate at
37°C with orbital shaking at 200 rpm.
2. Monitor the D600nm and when it reaches 0.6 induce protein expression by adding
IPTG to give a final concentration of 0.6 mM. Incubate for a further 3 h under the
same conditions.
3. Harvest the cells at 9000x g for 20 min at 4°C using the SLC-6000 rotor. Cell pellets
can be stored frozen at -80°C before further processing if necessary.
4. If the cell pellets are small, complete transfer from the centrifuge bottles is helped
by resuspending them in 2 mL/g 20 mM Tris-HCl, 0.5 mM EDTA, pH 7.5, containing
10% glycerol. Larger cell pellets can be transferred undiluted using a spatula.
-45. Resuspend the cell pellets (4-5 ml buffer/g cells) in ice-cold 20 mM Tris-HCl, 0.5
mM EDTA, pH 7.5, containing 1 tablet protease inhibitor cocktail without EDTA per
50 mL. Homogenise using a T18 basic ULTRA-TURRAX® homogeniser and then
disrupt by passing twice through a TS series continuous cell disruptor (Constant
Systems, UK) operating at 30 kPSI and 4°C.
6. Pellet the cell debris at 14000 x g for 45 min at 4°C using the SLA1500 rotor.
Carefully remove the supernatant and retain.
7. Collect the membranes by centrifuging the supernatant in a Beckman Optima L80
ultracentrifuge (Type 45 Ti rotor, 41,000 rpm i.e. ~131,500 gav, 2 h at 4°C).
If only mixed inner and outer membranes are required, resuspend in a minimal volume (a
few mL) of 20 mM Tris-HCl, 0.5 mM EDTA, pH 7.5 using a glass/Teflon homogeniser*.
Take a small sample for measurement of the protein concentration using the bicinchoninic
acid (BCA) assay, then snap-freeze the rest of the membranes in liquid nitrogen and store
at -80°C.
*Note, normally we use this method to produce mixed membranes prior to protein
purification by IMAC. In that case we resuspend the membranes to their original volume in
Tris buffer lacking EDTA and centrifuge again as in step 7 but for just 1 h in order to
remove the chelating agent before freezing, but EDTA can be left in if the membranes are
not to be used for such purifications.
If a purer preparation of inner membranes is required, proceed as follows:
Prepare sucrose gradients in 70 mL polycarbonate centrifuge tubes (for the Beckman
Type 45 Ti rotor) such that they contain the following layers from bottom to top of tube: 5
mL of 55% followed by 10 mL 50, 45, 40, 35 and 30% (w/w) sucrose in 20 mM Tris-HCl,
0.5 mM EDTA, pH 7.5.
To make the gradients, start with the least dense (30%). Using a 16 or 18 gauge needle
and a 10 mL syringe, pull 1 mL of air and then slightly more than 10 mL of the sucrose
solution into the syringe. Depress the plunger gently to the 10 mL mark to expel any air in
the needle, then remove any drops from the side and end of the needle by wiping against
the inside top of the stock sucrose container. Next gently expel the solution into the bottom
of the tube. Then successively add the denser solutions, starting with the 35% sucrose, by
injecting these carefully below the lowest layer using the syringe needle, thus displacing
the less dense layers. Cover the tubes with cling film to avoid contamination between
additions. Store in the cold room until required.
Resuspend the membrane pellet from step 7 in a small volume of 25% (w/w) sucrose in 20
mM Tris-HCl, 0.5 mM EDTA, pH 7.5, using a syringe if necessary to ensure a
homogenous suspension with no lumps. The volume of the resuspended pellet should not
exceed 6 mL per centrifuge tube.
Layer the membrane fraction onto the sucrose gradients. Each tube can accommodate a
maximum of 6 mL of the resuspended membranes. Balance the tubes using the
membrane suspension or some of the 25% (w/w) sucrose solution if necessary
-5Centrifuge in a Beckman Optima L80 ultracentrifuge (Type 45 Ti rotor, 38,000 rpm i.e.
~113,000 gav) at 4°C for 16 hours, using slow acceleration (9) and no brake.
Draw off the membrane layers, using separate syringes in order not to contaminate the
different membrane fractions. The golden inner membranes should be at the 35-40%
interface and the white outer membranes at the 50-55% interface.
Remove the sucrose by resuspending the membrane fractions in 20 mM Tris-HCl, 0.5 mM
EDTA, pH 7.5* then centrifuging as in step 7. Repeat twice to remove all traces of sucrose,
but reducing the centrifugation time to 1 h. Then snap freeze in Liquid Nitrogen and store
at -80°C
*Note, normally we use this method to produce inner membranes prior to protein
purification by IMAC. In this case we wash with Tris buffer lacking EDTA so as to remove
the chelating agent, but EDTA can be left in if the membranes are not to be used for such
purifications.
References
[1] Osborn, M.J., Gander, J.E., Parisi, E. & Carson, J. (1972) Mechanism of assembly of the outer
membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer
membrane. J. Biol. Chem. 247, 3962-3972.
Bam HI (1)
Stu I (5634)
Pst I (5526)
NupC
Xba I (7)
SalI (13)
Sbf I (23)
Nco I (5043)
Pst I (23)
Eco RV (5008)
Sph I (29)
Stu I (4797)
Hin dIII (31)
rrnB T 1 and T2 transcription terminator
Eco RI (4543)
rbs
ptac
lac operator
-10 lacUV5
-35 trp
pGJ L16
5743 bp
lacIq
pBR322 ori
Eco RV (1908)
Sca I (2714)
Ampr
For expression of un-tagged NupC, we use the pTTQ18-derived plasmid, pGJL16, illustrated above.
The encoded sequence is as follows, differing from the wild-type protein only by replacement of
residues 2 and 3 (DR) by NS as part of the cloning procedure:
MNSVLHFVLALAVVAILALLVSSDRKKIRIRYVIQLLVIEVLLAWFFLNSDVGLGFVKGFSEMFEK
LLGFANEGTNFVFGSMNDQGLAFFFLKVLCPIVFISALIGILQHIRVLPVIIRAIGFLLSKVNGMG
-6KLESFNAVSSLILGQSENFIAYKDILGKISRNRMYTMAATAMSTVSMSIVGAYMTMLEPKYVVAAL
VLNMFSTFIVLSLINPYRVDASEENIQMSNLHEGQSFFEMLGEYILAGFKVAIIVAAMLIGFIALI
AALNALFATVTGWFGYSISFQGILGYIFYPIAWVMGVPSSEALQVGSIMATKLVSNEFVAMMDLQK
IASTLSPRAEGIISVFLVSFANFSSIGIIAGAVKGLNEEQGNVVSRFGLKLVYGSTLVSVLSASIA
ALVL
This protein has a theoretical Mr of 43405.6 but will migrate on SDS-PAGE with an apparent Mr of
~34,000. In IPTG-induced cultures, it should be expressed at levels up to 20% of the inner
membrane protein and so should be apparent as a distinct Coomassie blue-stained band absent from
non-induced cells. (We used to have an antibody against NupC, raised against an ovalbumin
conjugate of a synthetic peptide corresponding to residues 220-235 of the protein in rabbits and then
affinity purified on a peptide column, but I don’t think we have any left. So, Coomassie blue
staining is the best way of checking expression, if membranes from induced and non-induced
cultures are run side by side on the gel. The band will be clearest if inner membranes are prepared,
but should be visible in membranes produced by the water-lysis procedure).