Expression & Purification of CymA
Theo Laftsoglou
Astbury Centre for Structural Molecular Biology, University of Leeds
Contents
A.
B.
C.
D.
E.
F.
Expression
Lysis & Preparation of the membranes
Membrane solubilisation
Affinity chromatography
Final preparation
Recipes
A. Expression
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Transform S. oneidensis MR-1 cells with pTL01 (kanamycinR; encodes the 8xHisHRVsite-CymA protein construct) by electroporation.
Culture a single colony from a selective plate in 10 mL LB medium supplemented
with 50 µg/ml kanamycin overnight at 30 °C / 200 rpm.
In the late afternoon of the following day, inoculate 100 mL (in 250 mL conical flask)
2YT medium with 3% inoculum from the overnight culture, supplemented with
antibiotic as before, and incubate overnight at 30 °C / 200 rpm.
In the afternoon of the following day, inoculate 1 L (in 2 L conical flask) TB,
supplemented with antibiotic as before, with 3% inoculum from the second
overnight culture. Incubate at 30 °C / 200 rpm until it reaches OD600 ~ 0.7. Then,
induce with 1 mM arabinose and incubate for expression for 16 hrs at 30 °C / 180
rpm.
On the following day, harvest the cells by centrifugation at 6,000xg for 15 min at 4
°C.
B. Lysis & Preparation of the membranes
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Resuspend the cell pellet in the resuspension buffer (170 mL per pellet from 1 L
culture – approx. 39g wet cell mass), supplemented with EDTA-free protease
inhibitors cocktail (Roche; per the manufacturers’ instructions).
Lyse the cells by passing them twice through the disruptor at 30,000 psi.
Centrifuge the lysate at 12,000xg for 50 min at 4 °C to pellet intact cells and cell
debris.
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Discard the pellet and centrifuge the supernatant at 100,000xg for 60 min at 4 °C to
pellet the membranes.
Discard the supernatant and homogenise the membranes in the resuspension buffer.
Centrifuge at 100,000xg for 60 min at 4 °C.
Discard the supernatant and homogenise the membranes in the resuspension buffer.
Perform the above steps that are highlighted in grey two more times. That is, for
washing of the membranes.
Homogenised membranes can be flash-frozen with N2(liq), and stored in -80 °C.
C. Membrane solubilisation
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Homogenise the membrane pellet in solubilisation buffer (12.5 mL per membranes
from 1 L culture) for 3 hrs at 4 °C.
Centrifuge at 100,000xg for 60 min at 4 °C to pellet insoluble material. The
supernatant comprises the column load.
D. Affinity chromatography
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Pre-equilibrate a 5mL-HiTRAP-HP column (or similar) with 20 CV of equilibration
buffer.
Load the column-load onto the column at 0.5 mL/min.
Wash the column with 10 CV of equilibration buffer.
Perform a second wash with 20 CV of 50 mM imidazole, or initiate an imidazole
gradient for elution (5 mM – 500 mM).
For step-elution, elute with 300 mM imidazole until there are no more red eluates.
Verify with SDS-PAGE, and pool the desire eluates.
E. Final preparation
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Dialyse the pooled eluates against the dialysis buffer for 2hrs at 4 °C.
Change the dialysis buffer and further dialyse overnight at 4 °C.
The next day, concentrate the protein, flash-freeze it with N2(liq), and store at -80 °C.
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F. Recipes
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LB medium (per 100 mL)
1 g Tryptone
1 g NaCl
0.5 g Yeast extract
-> autoclave
2YT medium (per 100 mL)
1.6 g Tryptone
1 g Yeast extract
0.5 g NaCl
-> autoclave
TB medium (per 900 mL)
12 g Tryptone
24 g Yeast extract
4 mL Glycerol
-> autoclave
Make separately 100 mL of 0.17 M KH2PO4, 0.72 M K2HPO4, and autoclave. (This
solution is made by dissolving 2.31 g of KH2PO4 and 12.54 g of K2HPO4). Finally, add
the phosphate buffer to the main solution under sterile environment.
Resuspension buffer
100 mM Hepes pH 7.5
100 mM NaCl
Solubilisation buffer
20 mM Tris •Cl pH 8.0
0.5 M NaCl
5 mM Imidazole
20% v/v glycerol
1% w/v DDM
Equilibration buffer
20 mM Tris ·Cl pH 8.0
0.5 M NaCl
5 mM Imidazole
20% v/v glycerol
0.1% w/v DDM
Elution buffers
20 mM Tris ·Cl pH 8.0
0.5 M NaCl
5 mM Imidazole
20% v/v glycerol
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0.1% w/v DDM
Imidazole to the desired concentration
Dialysis buffer
20 mM Hepes pH 7.5
150 mM NaCl
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