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Optimization of membrane protein overexpression and
purification using GFP fusions
David Drew1, 4, Mirjam Lerch1, 4, Edmund Kunji2, Dirk-Jan
Slotboom3 & Jan-Willem de Gier1
1 Department of Biochemistry and Biophysics, Stockholm
University, SE-106 91 Stockholm, Sweden.
2 MRC Dunn Human Nutrition Unit, Hills Road, CB2 2XY
Cambridge, United Kingdom.
3 Department of Biochemistry, University of Groningen,
Nyenborg 4, 9747 AG Groningen, the Netherlands.
4 These authors contributed equally to this work.
Correspondence should be addressed to Jan-Willem de Gier
degier@dbb.su.se
Optimizing conditions for the overexpression and purification
of membrane proteins for functional and structural studies is
usually a laborious and time-consuming process. This process
can be accelerated using membrane protein–GFP fusions1, 2, 3,
which allows direct monitoring and visualization of membrane
proteins of interest at any stage during overexpression,
solubilization and purification (Fig. 1). The exceptionally
stable GFP moiety of the fusion protein can be used to detect
membrane proteins by observing fluorescence in whole cells
during overexpression, with a detection limit as low as 10 mug
of GFP per liter of culture, and in solution during
solubilization and purification. Notably, the fluorescence of
the GFP moiety can also be detected in standard SDS
polyacrylamide gels with a detection limit of less than 5 ng
of GFP per protein band (Fig. 2). In-gel fluorescence allows
assessment of the integrity of membrane protein–GFP fusions
and provides a rapid and generic alternative for the
notoriously difficult immunoblotting of membrane proteins.
With whole-cell and in-gel fluorescence the overexpression
potential of many membrane protein–GFP fusions can be rapidly
assessed and yields of promising targets can be improved. In
this protocol the Escherichia coli BL21(DE3)-pET system—the
most widely used (membrane) protein overexpression system—is
used as a platform to illustrate the GFP-based method. The
methodology described in this protocol can be transferred
easily to other systems.
Figure 1. Flowchart illustrating optimization of membrane
protein overexpression and purification using GFP fusions.
Figure 2. Monitoring overexpression of membrane protein GFP
fusions using whole-cell and in-gel fluorescence.
(a) Indicated amounts of purified GFP-8His were run on a 12%
SDS polyacrylamide gel. In-gel fluorescence was monitored
(Steps 11–13) and then the gel was stained with Coomassie
(left). Intensities of in-gel fluorescent signals after 0.5 s
exposure were plotted versus the amounts of GFP-8His loaded
(right). (b) A culture of BL21(DE3)pLysS cells harboring
pYedZ-TEV-GFP-8His was grown as described in Steps 3–6. After
induction of expression of the YedZ-TEV-GFP-8His fusion with
0.4 mM IPTG at 25 °C, samples were collected at indicated
times. YedZ-TEV-GFP-8His expression was monitored by means of
whole-cell fluorescence (Steps 7–9) and in-gel fluorescence
(top; Steps 10–13). The whole-cell fluorescence signals were
plotted versus the intensities of the in-gel YedZ-TEV-GFP-8His
fluorescence signals (bottom). (c) To compare protein
production in different culture volumes, 13 different membrane
protein–GFP fusions were expressed in BL21(DE3)pLysS cells in
1-ml and 1-l cultures as described in Steps 3–6. Four hours
after induction of expression, whole-cell fluorescence was
monitored as described in Steps 7–9. The whole-cell
fluorescence of cells from 1-ml cultures was plotted versus
the whole-cell fluorescence of a 1-ml sample from the 1-l
cultures. MW, molecular weight.
MATERIALS
Reagents
1,4-dithiothreitol (DTT; Sigma)
Buffer A: phosphate-buffered saline (PBS) with 0.1%
n-dodecyl-beta-D-maltopyranoside (DDM; or other detergent at
5times critical micellar concentration; see Supplementary
Table 1 online)
Buffer B: Buffer A with 500 mM imidazole
Deoxyribonuclease I from bovine pancreas Type IV lyophilized
powder (Sigma)
E. coli BL21(DE3)–derived host strains (see Supplementary
Table 2 online)
Ethylenediaminetetraacetic acid (EDTA; Sigma)
Purified GFP (Supplementary Methods online)
Solubilization buffer (SB): 200 mM Tris-HCl (pH 8.8), 20%
Glycerol, 5 mM EDTA (pH 8.0), 0.02% bromphenol blue, make
aliquots of 700 mul and keep at -20 °C. Before use, add 200
mul 20% SDS and 100 mul 0.5 M DDT
Tobacco etch virus (TEV) protease, His-tagged (see
Supplementary Data online)
Equipment
1.5-ml polyallomer microcentrifuge tubes (Beckman)
ÄKTAprime or higher Äkta system (GE Healthcare)
Beckman TLA100 bench-top ultracentrifuge equipped with Beckman
TLA100 rotor
Centricon Centrifugal Filter Unit (Millipore); cutoff 30,000,
50,000 and 100,000 nominal molecular weight limit (NMWL)
depending on size of protein and detergent
LAS-1000 charge-coupled device (CCD) camera system (Fujifilm)
Nunc 96-well optical bottom plate, black (Nunc)
Poly-Prep chromatography columns (Bio-Rad)
Shaking incubator with temperature control
SpectraMax Gemini EM microplate spectrofluorometer (Molecular
Devices)
Superdex 200 10/300 GL Tricorn gel filtration column (GE
Healthcare)
Thermomixer comfort (Eppendorf) equipped with thermoblocks for
2.0-ml or 1.5-ml microcentrifuge tubes
Tunair 2.5-liter baffled shaker flasks
Ultracentrifuge, for example Beckman Coulter Optima LE-80k
equipped with Beckman Ti 70.1 rotor
Ultraviolet-visible (UV-Vis) spectrophotometer, for example
UV-1601 (Shimadzu)
XK 16/20 column (GE Healthcare) or larger column
Additional reagents are listed in Supplementary Methods.
PROCEDURE
Construction of genes encoding membrane protein–GFP fusions
1. Before cloning the genes encoding the membrane proteins
into the GFP-fusion vector, verify that the C termini of the
membrane proteins are in the cytoplasm (Cin topology; Fig. 1).
In E. coli, the GFP moiety of a membrane protein–GFP fusion is
fluorescent only if the fusion is integrated into the
cytoplasmic membrane (that is, inclusion bodies are not
fluorescent1, 3, 4) and has a Cin topology2. If the location
of the C terminus of the membrane protein to be overexpressed
is unknown, predict its topology using, for example, the
online application TMHMM
(http://www.cbs.dtu.dk/services/TMHMM). Approximately 80% of
all helical membrane proteins have a cytosolic C terminus5, 6,
7.
2. For each membrane protein, clone the gene of interest into
a standard pET28a(+)-derived GFP-8His fusion vector1, 8 that
harbors a TEV protease recognition site for removal of the
GFP-8His moiety during purification (see Supplementary Fig. 1
online). Note that we also use the abbreviation TEV to
indicate the TEV protease recognition site between the
membrane protein and GFP moiety; membrane
protein–TEV-GFP-8His.
A library covering the vast majority of E. coli membrane
proteins fused to GFP is available7.
Determining the overexpression potential of membrane
protein–GFP fusions
3. Transform the expression vector encoding a membrane
protein–GFP fusion into BL21(DE3)pLysS cells (see
Supplementary Methods). Use a fresh colony of the transformed
strain to set up an overnight culture in a 2-ml standard
microcentrifuge tube containing 1 ml LB medium with 50 mug/ml
kanamycin and 34 mug/ml chloramphenicol. Also set up a culture
to be used as control in Step 9 to measure background
fluorescence. This control can be a culture harboring the
expression vector that will be uninduced in Step 6 or a
culture harboring an 'empty' expression vector.
To target the most promising candidates, overexpression of
several membrane proteins as GFP fusions can be tested
simultaneously (Steps 3–13; Fig. 3a). Alternatively,
24-deep-well microtiter plates can be used for cultures in
Steps 3–6, although their handling is more cumbersome.
Figure 3. Examples of method application.
Figure 3 thumbnail
(a) Seven membrane protein–GFP fusions were screened for
overexpression in the strain BL21(DE3)pLysS by means of
whole-cell and in-gel fluorescence. Before loading onto gel
the cell suspensions were twofold concentrated for hKDELr and
twofold diluted for YciS. As controls, purified GFP-8His and a
sample of uninduced cells (YciS, fivefold concentrated) were
also loaded (U). (b) Whole-cell fluorescence from cells
overexpressing YbaT-TEV-GFP-8His was monitored after 4 and 22
h of expression. Colored bars represent the different strains
used: C41(DE3), green; C43(DE3), red; and BL21(DE3)pLysS,
blue. IPTG induction: 0.1 mM (odd numbers) or 0.4 mM (even
numbers). Error bars represent minima and maxima from three
independent experiments carried out in duplicate. (c) Protein
was detected by in-gel fluorescence on a 12% SDS gel. Numbers
correspond to the numbers in b. (d) Screening different
detergents for their efficiency to solubilize
YciS-TEV-GFP-8His–containing membranes. Error bars represent
minima and maxima from three independent screens. Inset,
in-gel fluorescence of solubilized material. LDAO,
lauryldimethylamine oxide; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
(e) Purification of YedZ-TEV-GFP-8His fusion and recovery of
YedZ from the fusion as analyzed after 12% SDS-PAGE by in-gel
fluorescence (left) and Coomassie staining (right). The lanes
were loaded as follows: 1, YedZ-TEV-GFP-8His–containing
membranes (12 mug); 2, non–detergent-solubilized protein (12
mug); 3, solubilized protein (12 mug); 4, IMAC flowthrough (4
mug); 5, YedZ-TEV-GFP-8His eluate from IMAC (2 mug); 6,
purifed YedZ-TEV-GFP-8His TEV digest (2.5 mug; note that
GFP-8His and YedZ are not separated by this percentage of
SDS-gel); 7, YedZ after gel filtration and removal of
His-tagged TEV protease and GFP-8His by batch-binding to
Ni-NTA resin; 8, GFP-8His (0.5 mug); 9, His-tagged TEV (0.5
mug). MW, molecular weight.
4. Incubate the culture overnight in a thermomixer at 37 °C at
900 r.p.m.
5. Dilute the overnight culture 50-fold into two 2-ml standard
microcentrifuge tubes, each containing 1 ml LB medium with 50
mug/ml kanamycin and 34 mug/ml chloramphenicol.
6. Incubate the cultures in a thermomixer at 900 r.p.m. at 37
°C and designate one of the tubes to monitor the optical
density (OD)600 of the culture. At an OD600 of 0.4–0.5 (after
approx2 h) lower the temperature to 25 °C and induce
expression of membrane protein–GFP fusion in the remaining
tube with isopropyl beta-D-thiogalactopyranoside (IPTG; 0.4 mM
final concentration).
Critical step
7. Four hours after induction, centrifuge the cells at 15,700g
for 2 min in a benchtop centrifuge, remove the supernatant
carefully and resuspend the cell pellet in 200 mul of PBS.
8. Transfer 100 mul of cell suspension to a black Nunc 96-well
optical-bottom plate. (Set aside the remaining 100 mul of cell
suspension for monitoring in-gel fluorescence in Steps 10–13).
9. Measure GFP fluorescence emission at 512 nm and excitation
at 485 nm in a microplate spectrofluorometer. Select the
option 'bottom read' for maximal sensitivity. Estimate
membrane protein overexpression levels (in mg/l; see
Supplementary Methods and Supplementary Fig. 2 online).
To assess background fluorescence levels in the system used,
measure whole-cell fluorescence of an uninduced sample or
cells containing an empty expression vector. Using the
microplate spectrofluorometer described here, fluorescence
counts for cells grown to an OD600 of 1.5 are twice that of
PBS.
Troubleshooting
10. Centrifuge the 100-mul cell suspension (set aside in Step
8) in a bench-top centrifuge at 15,700g for 2 min and remove
the supernatant carefully.
Pause Point The cell pellets can be stored at -20 °C for
several days.
11. Based on the whole-cell fluorescence measurement (Step 9),
resuspend the pellets in a volume of PBS to give a GFP
fluorescence level equal to that of 5–10 ng/mul of purified
GFP-8His (see Supplementary Methods). Add 10 mul of SB to 10
mul of each cell suspension and to 10 mul of purified GFP at a
concentration of 5–10 ng/mul. Incubate the samples at 37 °C
for 5 min.
Whole-cell fluorescence in the cell suspensions should be
adjusted to roughly similar levels to ensure that in SDS-PAGE
(Step 12) weak bands (that is, in case of degradation) can be
detected without interference of a much stronger signal in the
neighboring lanes. The most important advantage of using
in-gel fluorescence is to verify that full-length protein is
present. Quantification is also possible, but measuring
fluorescence in solution is less time-consuming.
Critical step
12. Analyze the samples from Step 11 by SDS-PAGE; include a
molecular weight marker.
13. Rinse the gel with distilled water. To detect the
fluorescent bands, expose the gel to ultraviolet light and
capture images with a CCD camera system (Fig. 3a). Increase
exposure time to desired band intensity.
Fluorescence intensities can be quantified using Image Gauge V
3.45 software or comparable software (Fig. 2a,b). If desired,
the gel can be subsequently stained with Coomassie.
Troubleshooting
Optimization of overexpression of membrane protein–GFP fusions
14. Transform the expression vector encoding a membrane
protein–GFP fusion selected from previous screen into
BL21(DE3)pLysS, C41(DE3) and C43(DE3) strains (see
Supplementary Methods and Supplementary Table 2).
These three strains give consistently good membrane protein
overexpression yields in our laboratory. The strain that gives
the best results for a particular membrane protein, however,
must be determined empirically.
15. Set up overnight cultures using fresh transformants in
standard 2-ml microcentrifuge tubes containing 1 ml of LB
medium with appropriate antibiotic(s).
Critical step
16. Incubate overnight cultures in a thermomixer at 37 °C at
900 r.p.m.
17. Dilute overnight cultures 75-fold into four 50-ml Falcon
tubes per strain, each tube containing 15 ml of LB medium with
appropriate antibiotic(s), and incubate at 37 °C at 220 r.p.m.
18. Monitor the OD600 of the cultures, and upon reaching an
OD600 of 0.25–0.35 (after approx2 h) shift the incubation
temperature for two cultures to 30 °C and, for the other two
cultures, to 25 °C.
Critical step
19. Grow the cultures at the lower temperatures for 30 min;
then induce expression of the membrane protein–GFP fusion by
adding IPTG. For each set of duplicate cultures grown at 30
°C, add IPTG to one culture to a final concentration of 0.1
mM, and, to the other culture, to a final concentration of 0.4
mM. Similarly, for each set of duplicate cultures grown at 25
°C, add IPTG to one culture to a final concentration of 0.1
mM, and to the other, to a final concentration of 0.4 mM.
There are now 12 different conditions represented (host
strain, temperature shift and IPTG concentration) as
summarized below.
20. Grow the strains in the presence of IPTG for 4 h, then
remove 1 ml of culture for whole-cell fluorescence
measurements (see Steps 7–9). Incubate the remaining cultures
overnight and repeat the whole-cell fluorescence measurements
after approx22 h (Fig. 3b).
OD600 and in-gel fluorescence can be monitored as well (Fig.
3c). The optimization screen is done in 50-ml Falcon tubes
instead of 2-ml tubes to provide enough volume for two
measurements (at 4 h and 22 h). Furthermore, optimizing
overexpression in 2-ml microcentrifuge tubes is unreliable for
the overnight estimates because of oxygen depletion.
Troubleshooting
Isolation of membranes
21. Select the strain that gives the best overexpression as
established by the overexpression optimization screen (Steps
14–20), and set up an overnight culture in a 200-ml shaker
flask containing 20 ml LB medium with appropriate
antibiotic(s) (see Supplementary Table 2).
22. Transfer the overnight culture into 1 l of LB medium with
appropriate antibiotic(s) in a 2.5-liter baffled shaker flask.
Incubate the culture at 37 °C at 220 r.p.m. and use the
parameters established in the overexpression optimization
screen to overexpress the membrane protein–GFP fusion (Fig.
2c). Before collecting the cells, remove a 1-ml sample for
measuring whole-cell fluorescence.
OD600 and in-gel fluorescence can be monitored as well. The
volume of the overnight culture in an appropriate shaker flask
depends on the number of 1-l cultures to be inoculated (use 20
ml per liter).
23. Collect the cells by centrifugation at 6,200g at 4 °C for
15 min. Decant the supernatant and resuspend the cell pellet
in 500 ml ice-cold PBS.
From this step on, even when not indicated, material should be
kept on ice or at 4 °C.
24. Centrifuge the resuspended cells at 6,200g at 4 °C for 15
min and decant supernatant. Resuspend the cell pellet in 10 ml
ice-cold PBS.
Pause Point Cell suspensions can be rapidly frozen in liquid
nitrogen and stored at -80 °C for up to 6 months. Use
screw-capped tubes for storage.
25. Add Pefabloc SC (1 mg/ml final concentration), DNase
(20–100 U/ml final concentration) and MgCl2 (1 mM final
concentration) and break the cells with a French press at
10,000 p.s.i. for at least two passes at 4 °C. Most cells are
broken when the suspension has turned from turbid to
transparent.
Alternatively, other methods of cell disruption can be
applied, such as sonication in combination with EDTA-lysozyme
treatment, homogenization and cell disruption using disruptors
from Constant Systems.
26. Remove the unbroken cells and debris by centrifugation at
24,000g at 4 °C for 12 min and collect the supernatant
containing the membranes. Repeat this centrifugation step to
clear the supernatant of any residual cells and debris.
27. To collect the membranes, centrifuge the cleared
supernatant at 150,000g at 4 °C for 45 min. Remove the
supernatant and resuspend the pellet in 10 ml ice-cold PBS
using a disposable 10-ml syringe with a 21-gauge needle. Fill
centrifugation tubes with ice-cold PBS to avoid collapsing of
tubes during ultracentrifugation in Step 28.
28. Collect the membranes by repeating centrifugation at
150,000g at 4 °C for 45 min. Resuspend the pellet-containing
membranes in 5 ml ice-cold PBS as described in Step 27, and
measure total amount of protein in the membrane suspension
using the BCA (bicinchoninic acid) protein assay kit.
If any EDTA was used in Step 25, it will be washed away and
will not interfere with immobilized metal ion affinity
chromatography (IMAC) in Step 36.
Pause Point Membrane suspensions can be rapidly frozen in
liquid nitrogen and stored at -80 °C for up to 6 months. Note,
however, that some membrane protein crystallographers avoid
freezing and storing membranes and continue with purification
immediately.
Troubleshooting
Detergent screen
29. Adjust the membrane suspension to a protein concentration
of 3.75 mg/ml. Transfer 800-mul aliquots of the suspension
into 1.5-ml polyallomer microcentrifuge tubes.
30. Select a range of different types of detergents to test
for membrane solubilization (see Supplementary Table 1). Add
200 mul of a selected detergent in PBS to each of the 1.5-ml
tubes containing 800 mul of membrane suspension. See
Supplementary Table 1 for the final percentage of each
detergent (the final protein concentration is 3 mg/ml).
Incubate the mixtures at 4 °C for 1 h with mild agitation.
31. Centrifuge the nonsolubilized material in a bench-top
ultracentrifuge at 100,000g at 4 °C for 45 min. Collect the
supernatant and measure GFP fluorescence in 100 mul of the
supernatant containing the solubilized membrane protein to
estimate the solubilization yields (see Supplementary Methods
and Fig. 3d).
GFP fluorescence changes maximally plusminus3% in the presence
of the detergents tested. The integrity of extracted membrane
protein–GFP fusions can be analyzed with the in-gel
fluorescence assay as described in Steps 11–13 (Fig. 3d). The
percentage of detergent solubilization can be estimated by
comparing the fluorescence in the detergent-solubilized
membranes to that of the fluorescence left in the
nonsolubilized pelleted membranes resuspended in the same
volume of buffer.
Troubleshooting
32. Determine the optimal protein:detergent ratio by repeating
Steps 29–31 with the most effective detergent (as established
in Step 31), at a constant percentage with increasing amounts
of protein (that is, 3–10 mg/ml protein).
Establishing the point at which an increase in protein still
yields a linear increase in GFP fluorescence (optimal
protein:detergent ratio) is important for enriching the
solubilized membranes with the membrane protein–GFP fusion.
Purification of membrane protein–GFP fusions
33. Using the protein:detergent ratio established in Step 32,
solubilize the membranes for purification by incubating the
membrane-detergent mixture at 4 °C for 1 h with mild
agitation.
34. Remove the unsolubilized material by centrifugation at
100,000g at 4 °C for 45 min. Remove a 200-mul sample of the
supernatant and measure fluorescence as described in Step 31.
Set aside the remaining 100 mul for subsequent analysis of the
purification by SDS-PAGE as described in Step 43 (Fig. 3e).
35. Pack an XK 16/20 column, using approx1 ml of Ni-NTA resin
per milligram of membrane protein–GFP fusion to be purified,
and equilibrate the Ni-NTA column with five column volumes of
Buffer A.
36. Add imidazole (10 mM final concentration) to the
solubilized membranes (supernatant from Step 34) and load onto
the Ni-NTA column at a slow flow rate (0.3–0.5 ml/min).
37. Wash the column with approx20 column volumes of 4% Buffer
B at a flow rate of 1 ml/min.
38. Deliver a gradient of 4–25% Buffer B over 20 column
volumes at a flow rate of 1 ml/min and collect fractions.
The fraction volume to be collected is proportional to the
size of the column; for example, for a 5-ml column usually
1-ml fractions are collected. Once the wash and elution
conditions have been established, step gradients can be used
instead of continuous gradients: wash the column with 20
column volumes at 2% less than the highest percentage of
Buffer B at which protein was still bound to the column.
39. Elute the fusion protein with 50% Buffer B at a flow rate
of 1 ml/min and collect all fractions. Save 100-mul samples
from the flowthrough, wash and elution fractions.
40. Measure GFP emission (see Steps 8 and 9) in the different
fractions and determine the amount of membrane protein–GFP
fusion (see Supplementary Methods). Estimate any losses in
each step (for example, in flowthrough).
The amount of fusion in the eluate should be determined by
measuring the GFP fluorescence, as the BCA assay measures
total protein (including contamination) and is affected by
cross-reactivity with imidazole at concentrations >50 mM.
Troubleshooting
Removal of GFP moiety from the membrane protein fusion
41. Add equimolar His-tagged tobacco etch virus (TEV) protease
to membrane protein–GFP fusion and adjust DTT and EDTA to a
final concentration of 1 and 5 mM, respectively and incubate
at 4 °C for 10 h or overnight (see Supplementary Data).
For commonly used detergents, such as
n-dodecyl-beta-D-maltopyranoside (DDM) and Triton X-100,
equimolar amounts of TEV protease typically suffice for a
complete overnight digest at 4 °C. Using small amounts of
membrane protein–GFP fusion and TEV or any other site-specific
protease, optimal cleavage conditions can be identified
rapidly with in-gel fluorescence.
42. Measure total protein concentrations in the different
fractions with the BCA assay for SDS-PAGE analysis (Step 43).
43. Analyze the solubilzed membranes (Step 34), IMAC
flowthrough (Step 36), wash fractions (Steps 37–38), eluate
(Step 39) and TEV-digest reactions (for completeness of the
digest; Step 41) by SDS-PAGE, adding an appropriate amount of
protein in a 10-mul volume to 10 mul of SB. Process as
described in Steps 12–13.
Troubleshooting
44. If digest is complete (from Step 41), concentrate it in
Centricon concentrators (the cutoff used depends on the size
of the protein).
45. Separate the proteins by standard gel filtration using a
Superdex 200 10/30 column. Remove 100 mul from each of the
(different) protein absorbance peaks and process as described
in Steps 8–9 to establish which fractions contain GFP-8His.
Note that GFP-8His and His-tagged TEV have a similar retention
time.
46. If the membrane protein and GFP-8His are in the same
fractions, remove GFP-8His and His-tagged TEV by adding Ni-NTA
resin equilibrated in the buffer used for gel filtration. Use
approx1 ml of resin per 5 mg of total protein.
47. Transfer the mixture to an empty Poly-Prep chromatography
column and collect the flowthrough (membrane protein). Wash
with 3 column volumes to recover membrane protein remaining in
the dead volume. Process the different fractions as in Steps
12–13 (Fig. 3e).
Troubleshooting
TROUBLESHOOTING
Problem: Expression yields are less than 200 mug per liter of
culture.
[Step 9]
Solution: Improve signal to noise ratio by increasing the
amount of cells analyzed; set up 5-ml cultures and resuspend
the cell pellet in 100 mul of PBS for fluorescence
measurements.
Problem: There is severe proteolysis of membrane protein–GFP
fusion.
[Step 13]
Solution: Induce expression for 1–2 h at 37 °C or express
overnight at low temperature (20 °C) (Step 6).
Problem: Expression yields are low.
[Step 20]
Solution: Try expression at different temperatures (20 °C and
37 °C), in different media (for example, Terrific broth,
2times YT, minimal medium), in different strains (for example,
BL21-CodonPlus(DE3))9 or change to homologs of the protein.
Problem: The yield is less than 60 mg of total protein per
liter (indicative of poor cell breakage).
[Step 28]
Solution: Add EDTA (1 mM final concentration) and lysozyme
(0.5 mg/ml final concentration) to the cell suspension and
incubate for 15–30 min on ice before breaking the cells. If
cells are treated with EDTA and lysozyme before disruption, 2
mM MgCl2 rather than 1 mM MgCl2 should be used in Step 25.
Problem: There is severe proteolysis of membrane protein–GFP
fusion.
[Step 31]
Solution: Use commercially available protease inhibitor
cocktails rather than Pefabloc SC only in Step 25. Add ligand
to increase stability of protein.
Problem: Not all of the protein binds to the column as evident
from the presence of GFP fluorescence in the flowthrough (see
Step 43).
[Step 40]
Solution: Increase the bed volume and, if necessary, use a
column of a larger diameter (Steps 35–39).
Problem: There is degradation of the purified fusion.
[Step 43]
Solution: See Troubleshooting, Step 31. Do not freeze cells or
membranes.
Problem: There is nonspecific binding of membrane protein to
Ni-NTA resin.
[Step 47]
Solution: Add imidazole to a final concentration of 5–10 mM to
buffer used in batch binding (Step 46).
Top
CRITICAL STEPS
Determining the overexpression potential of membrane
protein–GFP fusions, Step 3 Always use freshly transformed
BL21(DE3)pLysS cells (that is, not older than 2–3 d) and
medium with freshly added antibiotics. Do not use glycerol
stocks of transformed BL21(DE3)pLysS cells as starting
material because this can cause significant losses in
expression levels10.
Determining the overexpression potential of membrane
protein–GFP fusions, Step 6 Because OD600 measurements are
highly dependent on the photospectrometer used, it is strongly
recommended that the same instrument is used for the different
experiments. Diluting samples to OD600 values less than 0.3
increases accuracy of OD600 measurements substantially.
Determining the overexpression potential of membrane
protein–GFP fusions, Step 11 Samples should be heated to 37 °C
rather than 95 °C. Heating membrane proteins at 95 °C often
causes aggregation, and GFP loses fluorescence after
incubation at 95 °C. If frozen cells are used for the in-gel
fluorescence assay, add MgCl2 (1 mM final concentration) and
DNase (1–5 U per 10 mul of cell suspension) and incubate for
15 min on ice before adding SB.
Optimization of overexpression of membrane protein–GFP
fusions, Step 15 Always use freshly transformed BL21(DE3)pLysS
cells (that is, not older than 2–3 d) and medium with freshly
added antibiotics. Do not use glycerol stocks of transformed
BL21(DE3)pLysS cells as starting material because this can
cause significant losses in expression levels10.
Optimization of overexpression of membrane protein–GFP
fusions, Step 18 For C41(DE3) and C43(DE3) the OD600 at
induction is crucial; significantly decreased overexpression
yields have been observed if cells continued to grow at 37 °C
to cell densities higher than 0.5 before induction. Expression
in BL21(DE3)pLysS, however, is less sensitive to variations in
the OD600 of induction (note the smaller error bars in
BL21(DE3)pLysS compared to C41(DE3) and C43(DE3) in Fig. 3b),
making this strain the vehicle of choice for initial
overexpression screening (Steps 3–13).
Comments
The GFP moiety of a membrane protein–GFP fusion can be used
for direct, rapid and quantitative detection of membrane
proteins during overexpression and isolation. In this protocol
the E. coli BL21(DE3)-pET system is used as a platform to
illustrate the method. It should be noted that the protocol
can be easily modified and extended: for example, different
strains, expression vectors, culture media, buffer systems for
protein isolation or other site-specific proteases can be
used. An important prerequisite for using this method in E.
coli is that the membrane protein should have a cytosolic C
terminus, as GFP can only fold and become fluorescent in the
cytoplasm2, 11. Fortunately, approx80% of the multispanning
membrane proteins have a cytosolic C terminus5, 6, 7. If
expression hosts other than E. coli (for example, Lactococcus
lactis3, Saccharomyces cerevisiae or Pichia pastoris12) are
used for the overexpression and isolation of membrane
protein–GFP fusions, the GFP variant most suited for that
particular host should be used.
Thus far, it has not been possible to predict how well a
membrane protein can be overexpressed7, making the development
of methods with which membrane protein overexpression can be
rapidly and accurately monitored a bare necessity to improve
the throughput of membrane protein research. The GFP moiety is
not only a time saver: the sensitivity and accuracy with which
it can be monitored both in solution and in gels makes it a
superior alternative to the notoriously unreliable and
time-consuming immunoblotting of membrane proteins.
Functional membrane proteins can be recovered easily from
membrane protein-GFP fusions by cleavage with a site-specific
protease3 (Fig. 3e). Finally, it should be noted that the
GFP-based methodology described in this protocol is also
applicable to soluble proteins fused to GFP.
Top
Example of application
We expressed seven different membrane proteins as GFP fusions
in the E. coli strain BL21(DE3)pLysS, and monitored their
expression and integrity using a combination of whole-cell and
in-gel fluorescence (Steps 3–13; Fig. 3a). All proteins are E.
coli cytoplasmic membrane proteins unless noted otherwise:
YedZ (putative integral flavocytochrome3), YciS (unknown
function), YbaT (putative amino acid transporter3), ProW
(component of high-affinity transport system for glycine,
betaine and proline), hKDELr (human KDEL endoplasmic reticulum
protein retention receptor), GlpT (glycerol-3-phosphate
transporter) and AmpG (involved in peptidoglycan recycling).
Note that membrane proteins usually run faster on SDS-PAGE
than expected, typically at approx70–85% of their expected
molecular weight. In the experimental setup used, GFP runs at
a molecular weight of 22 kDa rather than 28 kDa because it
remains properly folded.
As determined by in-gel fluorescence, GlpT is a membrane
protein that is not stably overexpressed; the full-length
protein (band marked with an asterisk) is only a minor
component. This is in keeping with published observations that
GlpT has to be overexpressed in the presence of its substrate
to prevent proteolytic cleavage13.
We were able to improve the overexpression yields of the
membrane protein YbaT more than 10-fold using an
experience-based matrix screen (varying parameters:
temperature, IPTG concentration, duration of overexpression,
host strain) as outlined in Steps 14–20 (Fig. 3b). To detect
degradation of overexpressed material, we loaded protein
corresponding to equal amounts of fluorescence and analyzed
the in-gel fluorescence from each culture condition by
SDS-PAGE, and subsequently exposed the gel to ultraviolet
light for varying amounts of time. To calculate the amount of
degradation, we also loaded two different amounts of GFP-8His
standard onto the same gel (in this example, <5% of total
fusion was degraded). It should be stressed that the
overexpression screen shown in Figure 3b,c is an example. It
is our experience that the behavior of a membrane protein in
the screen cannot be predicted, that is, the 'optimal
conditions' for overexpression of a particular membrane
protein must be established experimentally.
Once established, production of the membrane protein–GFP
fusion can be reliably scaled up (Steps 21–22 and Fig. 2c),
and membranes can be isolated (Steps 23–29). The fluorescence
from the GFP moiety can conveniently be used for screening the
solubilization efficiency of the membrane protein–GFP fusion
into different detergents as outlined in Steps 30–32. A
detergent screen for YciS-TEV-GFP-8His is illustrated in
Figure 3d. After spinning down the nonsolubilized material, we
calculated the solubilization efficiency (Step 31); in this
example, LDAO is 90% efficient (once the detergent is
selected, the best protein:detergent ratio can also be
determined as described in Step 32). We assessed the integrity
of the detergent-solubilized material by measuring in-gel
fluorescence (Fig. 3d).
Detection of in-solution and in-gel fluorescence as well as
visual detection is convenient during purification of membrane
protein–GFP fusions. Analysis of the purification of
YedZ-TEV-GFP-8His and the recovery of YedZ from the fusion by
SDS-PAGE combined with in-gel fluorescence and Coomassie
staining is illustrated in Figure 3e. We solubilized membranes
containing YedZ-TEV-GFP-8His fusion with 1% DDM, at a protein
concentration of 8 mg/ml with a solubilization efficiency of
72%. For IMAC, we washed the column with 10 column volumes of
10% Buffer B and eluted the fusion in a step gradient of 50%
Buffer B (we recovered 90% of starting material). We cleaved
off the GFP-8His moiety by overnight incubation with an
equimolar amount of His-tagged TEV protease at 4 °C and
further purified the protein by gel filtration. As the
TEV-protease and the clipped-off GFP are His-tagged, any
carryover contamination can be simply removed by batch-binding
isolated membrane protein to Ni-NTA resin (see Steps 46–47).
At every stage during the overexpression and isolation of a
membrane protein–GFP fusion it is possible to calculate the
amount of membrane protein by measuring the GFP fluorescence
and comparing it to a purified GFP-8His standard
(Supplementary Methods).
Note: Supplementary information is available on the Nature
Methods website.