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Innovations Forum: Optimal solubilization of membrane proteins

Optimal solubilization screening strategies


for GST-fusion membrane proteins
A-K. Lundbäck*,†,‡, L. Haneskog†, L. Andersson†, A. Heijbel†, L. Ingemarsson†, and D. Birse*,§,¶
* Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
† Amersham Biosciences AB, Uppsala, Sweden
‡ Department of Biosciences, Karolinska Institute, Huddinge, Sweden
§ Current Address: TGN Biotech Inc, Sainte-Foy, Québec, Canada
¶ Corresponding author: Darcy Birse, dbirse@tgnbiotech.com

The hydrophobic nature of membrane proteins generally makes them more difficult to
study than soluble proteins. Detergents are used for solubilization of membrane proteins
and must be present in all purification steps to avoid aggregation of the proteins. Selecting
detergent and optimizing solubilization conditions for a specific membrane protein is
time-consuming and expensive. In this study, a small-scale screening strategy for
defining optimal solubilization conditions for glutathione S-transferase (GST) fusion
membrane proteins was developed and applied.

Introduction must be active in the presence of the detergent used for solubiliza-
To date, few three-dimensional structures of membrane proteins have tion. To assay GST function, the activity of purified GST was screened
been elucidated. The importance of membrane proteins as candidate in the presence of selected detergents at various concentrations.
drug targets (e.g. receptors, transporters) in the pharmaceutical
GST catalyzes the conjugation of 1-chloro-2,4-dinitrobenzene
industry clearly indicates that production of membrane proteins is
(CDNB) with glutathione and produces a product that shows
becoming an increasingly important issue. Despite the pressure to
strong absorption at 340 nm (ε = 9.6 mM-1·cm-1). The enzymatic
systematically produce membrane proteins, purification methods
activity of GST was measured with the GST Detection Module
are far less developed than for soluble proteins.
(Amersham Biosciences) according to the manufacturer’s protocol.
Both expression and purification methods of recombinant membrane The sample and reaction buffer volumes were adjusted to a final
proteins are significantly more complex than for soluble proteins. volume of 250 µl to accommodate a 96-well microplate format.
The effect of detergents on the yield and activity of isolated Detergent stock solutions were made in PBS (phosphate buffered
membrane proteins emphasizes the need to evaluate several saline; 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4,
detergent classes. This need, together with the high cost of 2.7 mM KCl, pH 7.5) supplemented with 1 mM dithiothreitol (DTT).
detergents makes purification method development strategies The detergent was dissolved to a concentration defined as a function
more expensive and complex. of its critical micelle concentration (CMC). Different concentrations
of the detergents were assayed. The absorbance at 340 nm was
The ultimate goal of this study was to develop a method that can be monitored with a spectrophotometer every 10 s for 5 min.
used for screening different types and concentrations of detergents
during the development of a purification method for a membrane Cell culture and membrane preparation
protein. The screening method described here can be applied to a The putative E. coli potassium channel, ecoKch, was used as a
wide range of membrane proteins. model for the solubilization screening method. The ecoKch protein is
composed of six transmembrane helices and has a molecular weight
Methods of 46 000 (1, 2). The gene coding for the ecoKch protein was inserted
GST activity in the presence of detergents in a pGEX-6P-1 (Amersham Biosciences) vector to produce a GST-
Recombinant proteins tagged with GST can be captured by affinity fusion protein (GST::ecoKch). The protein was expressed as described
chromatography using Glutathione Sepharose™ 4B medium provided (3). Cells were lysed by freezing/thawing. Cell debris and unbroken
in the GST MicroSpin™ Purification Module (Amersham Biosciences). cells were removed by centrifugation at 800 × g for 10 min at 4 ˚C.
For the capture of GST-tagged membrane proteins, the GST moiety Membranes were isolated by centrifugation at 48 400 × g for
30 min at 4 ˚C.

10 Life Science News 15, 2003 Amersham Biosciences


Innovations Forum: Optimal solubilization of membrane proteins

Small-scale solubilization assays


0.3× CMC
For small-scale experiments, it was necessary to evaluate if the 0.16 2× CMC
maximum microcentrifuge speed gave sufficient clarification of

activity (A340/min*ml)
4× CMC
detergent-solubilized material. The membrane pellet was solubilized
0.12 10× CMC
1:10 (w/v) in 10× CMC octyl-β-D-glucoside (β-OG), which was
previously found to only partially solubilize GST::ecoKch. Samples
were clarified by centrifugation at 18 000 × g in a Sigma 2K15 0.08
centrifuge for either 30 min at room temperature or 60 min at 4 ˚C.
For direct comparison, samples were centrifuged at 48 400 × g for 0.04
30 min at 4 ˚C in a Sorvall SS-34 rotor (Sorvall Instruments, DuPont)
The efficiency of the centrifugations was then evaluated by
0
comparing the GST enzymatic activity of the supernatants.

e
0
S

8
20
M

yl
35

at

lat
10
AP

P4
2E
O

os
DD

ol
n

ij
β-

ho
X-

C1

N
CH

rc
ee

Br
Small-scale purification

ch

sa
yc
n
Tw

ito

ox
Solubilization effects were assayed with six detergents:

Tr

de
dodecyl-β-D-maltoside (DDM); β-OG; 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propane sulfonate (CHAPS); Tween™ 20; Fig 1. The effect of selected detergents at different concentrations on GST enzymatic
activity. The dotted line represents GST activity in the absence of detergent.
Triton™ X-100; and, lauroylsarcosine sodium salt (sarcosyl). The
membrane pellet was solubilized 1:10 (w/v) in 5% (w/v) detergent
solutions. After clarification, 500 µl of the supernatant was decanted for 30 min at room temperature as compared with 60 min at 4 ˚C.
and purified with the GST MicroSpin Purification Module at room This observation suggests that the speed and time for centrifugation
temperature according to the manufacturer’s protocol. The columns of the solubilized material can be reduced from 48 400 × g to
contain 50 µl beds of Glutathione Sepharose 4B. The GST::membrane 18 000 × g for 30 min, providing a considerable advantage for
protein was eluted with 500 µl PBS supplemented with 0.2% (w/v) the ease and speed of processing the samples for screening.
detergent and 10 mM reduced glutathione. GST activity was assayed Membranes were solubilized with six different detergents at
after all purification steps. 10× CMC, clarified by microcentrifugation, and purified on
SDS-PAGE analysis GST MicroSpin columns. The yield of GST-fusion protein in the
Samples were mixed with an equal volume of loading buffer solubilization and chromatographic steps was the highest with
(100 mM Tris-HAc, pH 7.5, 2% SDS, 20 mM DTT) and incubated at DDM and sarcosyl, judging from the SDS-PAGE analysis (Figs 2
room temperature for 30 min. The samples were loaded on an SDS and 3). Corresponding yields of GST enzymatic activity were
ExcelGel™ and run for 80 min on a Multiphor™ II Electrophoresis highest for DDM, although considerable activity was found for
System at 15 ˚C with the limiting settings 600 V, 50 mA, 30 W. β-OG, Triton X-100, and CHAPS (Fig 4). A large portion of the
The gel was stained with Coomassie™ Brilliant Blue. material solubilized in sarcosyl was unbound, passing in the flow-
through fraction during chromatography. This was most likely due
Results and discussion to the sarcosyl inactivating GST. However, residual protein was
The screening procedure is based on the binding activity of GST to bound to the column and eluted (Fig 3).
Glutathione Sepharose 4B media. To allow solubility screening of
detergents commonly used for membrane protein solubilization, the
Triton X-100

detergents must not affect the GST-binding activity. To quickly assess


Tween-20

sarcosyl
CHAPS

this, pure GST was incubated in the presence of the selected


β-OG
DDM

detergents and the enzymatic activity measured. GST activity


M

was not significantly affected by detergent or increased detergent


concentration (Fig 1) in most cases. However, deoxycholate, cholate, Mr
(× 103)
and sarcosyl decreased the enzymatic activity of GST. Sarcosyl
concentrations as low as 0.3× CMC (0.54 mM) decreased activity.
Surprisingly, LDAO (data not shown) and CHAPS gave increased GST 94
enzymatic activity with increasing detergent concentration. The ➞
presence of impurities in the detergent preparation may have 67
GST::ecoKch
interfered with the assays. Alternatively, these detergents may have protein
solubilized GST aggregates allowing for higher GST activity than 43 (Mr 72 000)
detected in the absence of detergents.
Solubilization of the membrane pellet in β-OG and centrifugation at
Fig 2. 7.5% SDS-PAGE gel of GST::ecoKch solubilized in six different detergents (indicated
18 000 × g or 48 400 × g for 30 min at room temperature gave above the lanes). M = Low molecular weight marker.
essentially the same GST activity in the supernatant. Minor GST
activity differences were observed in samples after centrifugation

Life Science News 15, 2003 Amersham Biosciences 11


Innovations Forum: Optimal solubilization of membrane proteins

DDM β-OG CHAPS Tween 20 Triton sarcosyl


X-100 1

M F E F E F E F E F E F E
Determine the detergent effect
on the enzymatic activity of
purified GST with the
Mr
CDNB assay
(× 103)
96-well microplate

94

67
2
43

Solubilize membranes in
Fig 3. 7.5% SDS-PAGE gel of GST::ecoKch solubilized in six different detergents and different detergents and
purified on GST MicroSpin columns. Detergents are indicated above the lanes. M = Low concentrations that do
molecular weight marker ; F = flowthrough; E = eluate. not affect the activity of
the GST tag

0.8 solubilized material


flowthrough
0.6 eluate
activity (A340/min*ml)

0.4 3

0.2
Purify fusion protein using GST
MicroSpin Purification Module

0
0
G

20
M

l
PS

y
10
O

os
DD

n
β-

X-
CH

rc
ee

sa
n
Tw

ito
Tr

Fig 4. The enzymatic activity of GST in six different detergents with compensation for
volume differences. Note that sarcosyl greatly decreases GST activity in CDNB assays,
and thus values do not represent the relative amount of GST fusion protein. Analyze yield by SDS-PAGE and
activity of membrane protein
(if assay is available)
Conclusion
When solubilizing a target membrane protein, several detergents Fig 5. Detergent screening strategy for a membrane protein fused to GST.
require evaluation prior to selecting the optimal detergent for
developing a purification strategy. This evaluation is often time- 3. Dian, C. et al. Strategies for the purification and on-column cleavage of
consuming and expensive. To minimize the loss of material when glutathione-S-transferase fusion target proteins. J.Chromatogr. B.
screening detergents, it is necessary to do small-scale experiments 769,133–144 (2002).
and to be able to process multiple samples in parallel, ideally in a
multi-well format. GST-membrane protein fusions make it possible
to monitor the presence of the fusion protein throughout the
Ordering Information
purification steps with the CDNB enzymatic assay. The method that
GST MicroSpin Purification Module (50 columns) 27-4570-03
was developed and applied (Fig 5) provides a simple and rapid method
GST Detection Module (50 detections) 27-4590-01
to select for optimal solubilization conditions to obtain the highest
pGEX-6P-1 (25 µg) 27-4597-01
yield of membrane protein with an active GST-fusion affinity tag.
Glutathione Sepharose 4B 27-4574-01
References (function tested) (100 ml)
1. Milkman, R. An Escherichia coli homologue of eukaryotic potassium channel
To obtain a brochure on the GST system, please circle 2 on the reader reply card, or
proteins. Proc. Nat. Acad. Sci. USA 91, 3510–3514 (1994).
download it at www.lsn-online.com/info.
2. Johansson, M. and von Heijne, G. Membrane topology of Kch, a putative K+
channel from Escherichia coli. J. Biol. Chem. 271, 25912–25915 (1996).

12 Life Science News 15, 2003 Amersham Biosciences

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