Membrane Protein Protocols ...

Membrane Protein Protocols [Methods In Molec Bio, Książki, Biochemia

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Methods in Molecular Biology
TM
VOLUME 228
Membrane
Protocols
Expression, Purification,
and Characterization
Expression, Purification,
and Characterization
Edited by
Barry S. Selinsky
Membrane
Protein
Protein
Protocols
Edited by
Barry S. Selinsky
1
Expression and Purification of the Amphipathic
Form of Rabbit Cytochrome b
5
in
Escherichia coli
Lucy Waskell
-helix
(1–3)
. The
membrane-bound form of cyt b
5
provides reducing equivalents for the biosyn-
thesis of a variety of lipids including unsaturated fatty acids, plasmalogens, and
cholesterol. In addition, it facilitates the cytochrome P450 catalyzed oxidation
of selected substrates
(2)
. The membrane domain is linked to the amino-
terminal catalytic heme-containing domain via an 11 amino acid linker. The
mammalian cyts b
5
are typically greater than 90% similar in sequence and may
be interchangeable in some systems
(4)
. Nevertheless, our laboratory uses rab-
bit cytochrome P450 2B4 and cytochrome P450 reductase and has elected to
use the rabbit cyt b
5
so all proteins are from a single species. Cyt b
5
also exists
in a soluble form in red blood cells where it functions to maintain hemoglobin
in its ferrous oxygen-carrying form
(5)
.
2. Materials
2.1. Escherichia coli
(E. coli)
Strains, Media, and Equipment
1. The key to the marked and reproducible overexpression of the membrane bound
form of cyt b
5
is use of the
E. coli
strain C41, a derivative of
E. coli
DE3 (Avidis
SA, Saint Beauzire, FR)
(6)
. This strain moderates the expression of genes down-
stream from a T7 promoter, thereby, decreasing the toxicity of the large amount of
mRNA generated
(7)
(Life Technologies-Gibco BRL) (
see
Note 1
).
From:
Methods in Molecular Biology, vol. 228:
Membrane Protein Protocols: Expression, Purification, and Characterization
Edited by B.S. Selinsky © Humana Press Inc., Totowa, NJ
3
1. Introduction
Cytochrome b
5
(cyt b
5
) is an electron transfer protein that exists in a
membrane-bound form in the endoplasmic reticulum where it is anchored to the
membrane via a carboxyl-terminal transmembrane
4
Waskell
g/mL (Life Technologies-Gibco RBL).
3. Luria Bertani (LB) medium: 10 g/L NaCl, 10 g/L peptone, 5 g/L yeast extract, 1
mL 1N NaOH.
4. Terrific broth (TB) medium: prepare by dissolving 12 g of bacto-tryptone, 24 g
bacto-yeast extract and 4 mL glycerol in 900 mL water. Sterilize for 20 min and
allow to cool. Immediately before use, add 100 mL of a sterile solution of 0.17
M
KH
2
PO
4
and 0.72
M
K
2
HPO
4
. If the TB medium is autoclaved in the presence of
phosphate buffer, a precipitate will occur.
5. Sterile filtered stock solution of 100 mg/mL carbenicillin made fresh prior to use.
6. 1
M
isopropyl-1-thio-
-D galactopyranoside (IPTG) in water. Store at
−
20°C.
20°C.
8. Equipment includes 2.8-L Fernbach flasks, Beckman JA10 rotor, Beckman J2-21
centrifuge (or equivalent), Vibra Cell sonicator (Sonic Materials) 3 mm and 1 cm
diameter probe, spectrophotometer (Cary 1, Cary 300 Bio or equivalent). Innova
Incubator Shaker 4430 (New Brunswick Scientific, Edison, NJ) or equivalent
shaker and autoclave.
-aminolevulinic acid (
-ALA). Store at
−
m filter. Store at 4°C.
2. Detergents: 10% (v/v) Tergitol NP-10 (Sigma). Store at 4°C. Solid Na deoxy-
cholate (Fisher Biotech).
3. Sodium hydrosulfite (sodium dithionite, [Sigma]).
4. BCA protein assay (Pierce).
5. Mini-Protease inhibitor tablets (Boehringer Mannheim).
6. All buffers should be filter sterilized using a 0.2
m filter.
7. Buffer A: 10 m
M
phosphate buffer, 1 m
M
ethylenediaminetetraacetic acid (EDTA),
pH 7.0.
8. Buffer B: 10 m
M
phosphate buffer, 1 m
M
EDTA, pH 7.0, 1% Tergitol NP-10.
9. Buffer C: 20 m
M
Tris-HCl, 1 m
M
EDTA, pH 8.0 at 25°C, 0.4% Na deoxycholate.
20 m
M
phosphate buffer can be used instead of Tris-HCl.
10. Buffer D: Buffer C, plus 0.4
M
NaCl.
11. Buffer E: 20 m
M
phosphate buffer, pH 8.0, 1 m
M
EDTA, 0.4% Na deoxycholate.
12. Buffer F: 50 m
M
Tris-acetate, pH 8.1 at 22°, 1 m
M
EDTA.
13. Resins: DEAE Sepharose Fast Flow (Sigma), Superdex-75 prep grade (Amersham
Pharmacia Biotech), Sephadex G-25 optional (Sigma).
14. Equipment includes chromatography columns from Bio-Rad, Foxy Jr., or Foxy 200
fraction collector (Isco, Lincoln, NE) or equivalent, Cary spectrophotometer.
2. LB Agar, 32 g/L water. Autoclave at 121°C for 15 min. Cool and pour into plates. Add
carbenicillin to a final concentration of 100
7. 200 mM
2.2. Reagents, Chromatography Resins,
and Equipment for Cyt b
5
Purification
1. A 1 m
M
solution of heme is prepared by adding hemin chloride to a solution of
50% ethanol in water and 0.1
N
NaOH. After the hemin chloride dissolves, filter the
solution through a 0.2
Rabbit Cytochrome b
5
in
E. coli
5
3. Methods
3.1. Expression of cyt b
5
1. C41 cells were transformed with the plasmid pLW01-b
5
mem using standard proce-
dures
(8
,
9)
. The transformed cells were plated from a 15% glycerol stock solution
onto a LB plate containing 100
-aminolevulinic acid and 250
M
and the cells were incubated with shaking for an additional 16–20 h.
5. Remove 2 mL of the culture and set aside for determination of cyt b
5
content (
see
later).
6. The remainder of the cell culture was chilled, poured into 500-mL plastic bottles
and centrifuged at 11,000
g
for 10 min at 4°C to pellet the whole cells. The cell cul-
ture was centrifuged in a JA10 rotor at 8000 rpm for 30 min at 4°C in a Beckman
J2-21 centrifuge.
7. Discard the supernatant and resuspend the cells in
25 mL of cold Buffer A.
8. Repellet the cells under the same conditions. An average of 8.8 g bright pink cell
paste is recovered from 500 mL cell culture (
see
Note 2
).
≅
3.2. Determination of the Amount of Apocyt b
5
in the Cell Culture and Reconstitution of Holocyt b
5
with Heme
1. Because most of the cyt b
5
is expressed as the apoprotein without the heme, it must
be reconstituted with heme prior to purification (
see
Note 3
). Centrifuge the 2 mL
aliquot of the cell culture at 10,000
g
for 1 min at room temperature.
2. Discard the supernatant and resuspend the pink cell pellet in 2 mL Buffer B.
3. Sonicate the cells using a Vibra Cell sonicator (Sonic Materials) with two 30 s pulses
at 40% power at 50 W. Immerse the cell suspension in a ice/water slush to keep the
temperature below 9.5°C. Cool to 4°C between each pulse. Be sure to sonicate vig-
orously enough to lyse all the cells. The heme cannot penetrate the bacterial cell
membrane and will not be able to reconstitute the apocyt b
5
within the cell. Incom-
plete reconstitution of the cyt b
5
will result in a poor yield.
4. Dilute the sonicated cells 20-fold with Buffer B and record the absorbance spec-
trum between 350–650 nm.
5. Add 2
≅
g/mL carbenicillin and incubated overnight at 37°C.
2. A single colony was picked and inoculated into a 2.8 L Fernbach flask containing
500 mL of TB medium supplemented with 0.5 m
M
g/mL carbenicillin.
3. The cultures were incubated at 37°C on an Innova Incubator Shaker 4430 (or
equivalent) with shaking at 140 rpm.
4. When the OD of the cultures was 0.35 at 600 nm, IPTG was added to a final concen-
tration of 10
1 m
M
heme solution to the sonicated cells and record the
spectrum after each addition. The difference spectrum (final spectrum-initial spec-
trum) should resemble the spectrum of cyt b
5
as long as the added heme is forming
holocyt b
5
(7)
. When the difference spectrum caused by addition of the heme
begins to resemble that of the heme and not cyt b
5
, the apocyt b
5
has been com-
L aliquots of a
6
Waskell
L aliquots of heme and plot the increase in absorbance at 412 nm.
When the apocyt b
5
is completely reconstituted, the absorbance increase at 412 nm
produced by a 2-
L heme aliquot will decrease, i.e., the slope of the line found by
A at 412 vs heme added will decrease. The point at which the change in
slope occurs indicates that the apocyt b
5
has been saturated with heme. Once the
amount of heme required to reconstitute the apocyt b
5
in a 2-mL sample is known,
the amount of heme necessary to reconstitute the apocyt b
5
in the 500 mL cell cul-
ture is readily determined. Reconstitute holocyt b
5
10%
molar excess of heme. If too much excess heme is added, it will be difficult to
remove and will interfere with quantitation of cyt b
5
.
with no more than a
≅
3.3. Measurement of Holocyt b
5
Holocyt b
5
is measured as previously described
(10)
. Briefly,
1. Place 1 mL of sample into both a reference and sample cuvet and record a baseline
in the spectrophotometer.
2. Reduce the cyt b
5
in the sample cell by addition of
1mgofsolid sodium dithionite.
3. Record a difference spectrum by subtracting the oxidized spectrum from the reduced
spectrum and determine the change in absorbance between 426 and 409 nm. Upon
reduction, cyt b
5
increases its absorbance at 426 nm and decreases its absorbance at
409 nm. An extinction coefficient of 185 m
M
−
1
cm
−1
for the absorbance change at
426 minus 409 nm was used to calculate the amount of cyt b
5
present. When the pro-
tein is pure and other interfering compounds are absent, an extinction coefficient of
117 m
M
−
1
cm
−1
at 413 nm was used to calculate the amount of cyt b
5
(11)
.
≅
3.4. Lysis of
E. coli
and Membrane Isolation
All of the following procedures with the exception of the chromatography on
DEAE were performed at 4°C (
see
Note 4
).
25 mL Buffer A.
2. Add two Mini-Protease inhibitor tablets and dissolve them in the cell paste (
see
Note 5
).
3. Sonicate the cells using a 1-cm-diameter probe that is immersed 2 cm into the cell
suspension. Sonicate the cells with
≅
6 pulses of 2-min duration at 80% power
using a 50-W setting. Immerse the cell paste in an ice/water slush during the soni-
cation and do not let the temperature rise above 9.5°C. Other equivalent methods of
cell lysis can be used. Regardless of which method of cell lysis is used, it is impor-
tant to ensure that all cells have been lysed in order to obtain a good yield.
4. Following cell lysis, reconstitute the apocyt b
5
by adding a 10% molar excess of
heme based on the amount of apocyt b
5
present in 2 mL of the lysed cell suspension.
5. Centrifuge the sonicated cells at 3000
g
for 15 min at 4°C to remove any unlysed
cells. If the pellet is red, resonicate to lyse any remaining intact cells.
6. Centrifuge the membrane containing supernatant at 100,000
g
for 1 h at 4°C.
≅
pletely converted to holocyt b
5
. An alternative procedure is to titrate the sonicated
cells with the 2
plotting
1. Defrost the pink cell pellet and resuspend in
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