Expression and purification of Nup internal His tag
Materials:























Bicinchoninic acid (BCA) reagent (Thermo, 23223)
Escherichia coli BL21Star™ (DE3) + pRARE2 competent cells (home made)
Carbenicillin (Melford, C0109)
Chloramphenicol (Alfa Aesar, B20841.22)
Agar (Oxoid, LP0011)
Glucose (Ducheta Biochemie, G0802.1000
His-Pur Co2+ resin (Pierce, 89965)
Imidazole (Acros, 122025000)
NaCl (Fisher, S/3160/63)
KH2PO4 (Melford, P0574)
IPTG (Generon, C9H1805S)
MES (FW 195.23) (Acros Organics, 172591000)
CaCl2 (Fisher, BP510-100)
Casamino acids (MP Biomidicals, 3060-012)
MgSO4x7H2O (BDH, 101514Y)
Na2HPO4 (Melford, S2002)
K2HPO4 (VWR, 26931.263)
n-dodecyl-β-D-maltoside (DDM), (Melford, B2010)
FOS-CHOLINE-14 (FC14), Affymetrix, F12)
NH4Cl (Melford, A2042)
Tryptone (Melford, T1332)
Vivaspin MWCO 100000 (Sartorius, VS0241)
Yeast extract (Melford, Y1333)
Stocks:











20x M9 (per L) autoclaved: 120g Na2HPO4, 60g KH2PO4, 20g NH4Cl, 10g NaCl
MgSO4x7H2O 1M autoclaved
CaCl2 0.1 M autoclaved
Glycerol 50 % autoclaved
Casamino 20% acids filtered and frozen as aliquot at -20C to prevent contamination
25% DDM (w:v) in water
Imidazole 3 M pH 7.4
KH2PO4 1M
K2HPO4 1M
KPi pH 7.4 (19 ml KH2PO4 + 81 ml K2HPO4)
MES 1M pH 6.8 frozen as aliquot at -20C to prevent photodegradation and
contamination
Media preparation:



Luria Bertani (LB)-medium (per L): 10 g tryptone, 5 g yeast extract, 10 g NaCl
LB-agar: LB supplemented with 1 % agar
M9; 0.1% Na2HPO4, 0.3% KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 0.2% casamino acids, 2
mM MgSO4, 0.2 mM CaCl2, 0.2% glycerol pH7
Expression and isolation of membranes:
Day 1:
 Transform E. coli strain BL21Star™ (DE3) (Invitrogen) harbouring the plasmid
pRARE2 (Novagen), with pLH13 (pTTQ18-based intra-His tagged NupC),
 Plate on LB-agar supplemented with carbenicillin (100 µg/ml) and chloramphenicol
(30 µg/ml) and incubate overnight at 37 °C.
Transformation protocol:
(4°C - ∞, 4°C – 30min, 42°C – 30sec, 4°C – 2min, 4°C - ∞, 37°C – 60min, 4°C - ∞)
Day 2:
 In the morning, inoculate 5 ml Luria-Bertani (LB) medium, supplemented with same
antibiotics, with a single colony and incubate at 37 °C with orbital shaking at 200
rpm for 6h.
 In the evening, inoculate two 250 ml flasks containing 50 ml of LB media,
supplemented with same antibiotics, with 1 ml cell culture.
Day 3:
 Inoculate 500 ml M9 medium ,supplemented with same antibiotics, into 2.5 L flask
at 0.05 DO with the overnight preculture.
 Incubate at 37 °C with orbital shaking at 200 rpm until D600 is about 0.6.
 Add IPTG for a final concentration of 1 mM (500 µL-1 M IPTG) and incubate for 4 h
at 37 °C with orbital shaking at 200 rpm.
 Several purifications can be conducted with a grow-up of 4 L. Alternatively prepare
30 litre culture and hand over the dry chemicals to David Sharples.
Day 4:
 Harvest the cells (9000xg 20min using SCL6000 rotor).
 Resuspend the cells (6 ml buffer/g cells) in 20 mM Tris, 0.5 mM EDTA pH 7.4
(measured at 4 °C) and PI tablets (one per 50ml)
 Lyse the cells at 30 kPSI using a cell disruptor (Constant Systems).
 Pellet the cell debris at 14000xg for 45min using SLA1500 rotor,
 Ultracentrifuge the supernatant at 100000xg (41k rpm) using Ti45 rotor for 2h at
4°C to collect membranes.
 Resuspend the membranes in the same volume in 20 mM Tris pH 7.4 (measured at
4 °C) and pellet again.
 Finally, resuspend the pellet in a small volume (typically ~ 3 ml) of 20 mM Tris pH
7.4 (measured at 4 °C) and snap-freeze in liquid nitrogen.
Determine the protein concentration in the membranes using a BCA assay.
Purification:
Dilute 25 mg of membrane protein to 5 mg/ml in 50 mM KPi pH 7.4, 150 mM NaCl, 5 mM
imidazole, 10% glycerol supplemented with 1 % DDM; incubate on a roller mixer for 1h at
4 °C.
Pellet the insoluble fraction in an ultracentrifuge at 100000g (41k rpm) for 1h at 4°C.
Pre-equilibrate 80 µl Co2+-resin slurry/mg initial protein with 50 mM KPi pH 7.4, 150 mM
NaCl, 5 mM Imidazole, 10% glycerol, 0.05 % DDM. Hereto, the slurry is first sedimented in
a 15 ml Falcon tube at 500xg for 3min, followed by washing with MilliQ H2O (15 ml) and
then an equal volume of buffer, sedimenting the resin between each washing. Then add
the supernatant to the resin and allow protein binding to occur overnight at 4 °C with gentle
mixing on a roller mixer.
At room temperature pack the resin into a column and wash with ~ 1 column volumes (CV
– calculated at 1 CV = 2 ml for this amount of resin) of 50 mM KPi pH 7.4, 150 mM NaCl,
5 mM Imidazole, 10% glycerol, 1 % FC14. Washing should be dropwise (1 drop/1-2 s)
under gravity flow.
Exchange the detergent by washing with 9 CV of 50 mM KPi pH 7.4, 150 mM NaCl, 15
mM imidazole, 10% glycerol, 0.05 % FC14.
Elute the protein using 5 CV 50 mM MES pH 6.8, 150 mM NaCl, 300 mM Imidazole, 10%
glycerol, 0.05 % FC14 at 4 °C. Elution again should occur dropwise in 500 µl fractions,
about 5 fractions. Fractions are evaluated for the presence of protein by measurement of
the A280. Pool the peak fractions. Usually 10 fractions are positice. Pool the peak positive
values as MAJOR and the flanking positive fractions as MINOR.
Dialyse the pooled fractions in 50 mM MES pH 6.8, 150 mM NaCl, 10% glycerol, 0.05 %
FC14 over night at 4 °C.
Dilute if necessary the pooled fractions by 2 with 50 mM MES pH 6.8, 150 mM NaCl, 10%
glycerol, 0.05 % FC14 and concentrate the protein to 5 mg/ml using a Vivaspin
concentrator with 100000 kDa cut-off by centrifugation at 3800 g at 4 °C.