From the
We have developed procedures to purify highly functional
recombinant cystic fibrosis transmembrane conductance regulator (CFTR)
from Chinese hamster ovary (CHO) cells to high homogeneity.
Purification of CHO-CFTR was achieved using a combination of alkali
stripping,
Cystic fibrosis (CF),
The best characterized
abnormality associated with CF cells is altered epithelial chloride
permeability. In consequence following cloning of the gene encoding
CFTR, it was initially suggested that CFTR was either a chloride
(Cl
The most common CF-associated mutation, a
deletion of the residue phenylalanine at position 508 (
Development of a
protein replacement therapeutic approach for CF requires that we
develop 1) an abundant source of the membrane protein, 2) processes for
purification of CFTR to homogeneity, and 3) a vehicle capable of
mediating the delivery of the reconstituted and purified CFTR to the
target cells. In this report, we describe procedures to purify
functionally active CFTR from CHO cell lines capable of producing high
levels of CFTR. The properties of the purified CHO-CFTR were studied
and compared using a variety of biochemical and electrophysiological
assays to CFTR purified from Sf9 cells using a modification of the
procedure described by Bear et al. (1992). The ability to
deliver CFTR to a target cell by membrane fusion has recently been
demonstrated (Marshall et al., 1994). Transfer of the crude
CFTR-containing membranes to the recipient cell was mediated by the
influenza virus hemagglutinin fusion protein. The feasibility of this
approach has now been extended to include the use of purified and
reconstituted CFTR (generated using the processes described here) in
either a proteoliposome or hybrid virosome.
Phosphatidylethanolamine, phosphatidylcholine (PC),
phosphatidylserine,
Insect Sf9 cells were infected with a recombinant baculovirus
containing the complete human CFTR cDNA. CFTR was purified from
infected insect cell membranes according to the method of Bear et
al.(1992), with the following modifications. A small 2.6
One-dimensional SDS-PAGE was performed using 4-20%
gradient (Daiichi) gels. Proteins in the gel were detected by staining
with either silver stain or Coomassie Blue. The purity of the CFTR
preparations was assessed by densitometry of the Coomassie-stained
SDS-PAGE gels using a LKB 2202 Ultroscan laser densitometer. For
immunoblotting, polyvinylidine difluoride membranes (Novex) were
prewetted with methanol and then soaked in 10 mM CAPS, pH 11,
containing 10% methanol. Gels were equilibrated in this transfer buffer
for 10 min and then blotted at 30 V for 3 h in a Novex blot module.
After transfer, membranes were blocked with 1% dried milk in
Tris-buffered saline (20 mM Tris-HCl, 150 mM NaCl, pH
7.5) for 1 h. After blocking, the membranes were probed with
biotinylated antibody (mAb 13-1 or mAb 24-1) at 0.05
µg/ml in 20 mM Tris, 150 mM NaCl, pH 7.5, and
0.05% Tween 20 containing 0.1% bovine serum albumin for 2 h. The
membranes were incubated with Streptavidin/horseradish peroxidase
(1:25,000) for 20 min, and the immunoreactive bands were visualized by
chemiluminesence using the ECL system (Amersham Corp.).
CFTR was quantified using an ELISA assay. ELISA plates were
coated with mAb 13-1 (Anti-R-domain antibody). Biotinylated mAb
24-1 (anti-C domain) and orthophenylene diamine (Engvall and
Perlman, 1971) were used as substrate to measure antigen specific
antibody response. Absorbance was measured at 490-650 nm.
10-µg (25-50 µl) samples of purified CFTR
(either in detergent or reconstituted in proteoliposomes) were
equilibrated at room temperature for 15 min with 100 µM
8-N
The cAMP-stimulated chloride channel activity of CFTR in both
insect and CHO cells was determined using the halide-sensitive
fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) as
described previously (Illsley and Verkman, 1987; Rich et al.,
1990; Cheng et al., 1991; Marshall et al., 1994). CHO
cells were loaded by hypotonic shock for 4 min at room temperature
while insect cells required a longer incubation time of 4 h for
effective loading. SPQ fluorescence was initially quenched by
incubating the cells for up to 30 min in a buffer containing 135 mM NaI, 2.4 mM K
Immunoaffinity-purified CFTR was concentrated as described
above and reconstituted using a modification of the procedure described
by Bear et al.(1992). An aliquot containing a known amount of
CFTR (15-20 µg) was added to 100 µl of 15 mM
Hepes, 0.5 mM EGTA, pH 7.4, containing 1 mg of a sonicated
phospholipid mixture
(phosphatidylethanolamine/phosphatidylserine/phosphatidylcholine/ergosterol,
5:2:1:2 (molar ratio)) and 1% sodium cholate. After a 40-min incubation
on ice, the mixture was dialyzed at 4 °C against 15 mM Hepes, 0.5 mM EGTA, pH 7.4, and 1.5% sodium cholate for
24 h. Dialysis was continued for an additional 3 days against 15 mM Hepes, 0.5 mM EGTA, pH 7.4, with daily changes of buffer.
The sample was further dialyzed against 15 mM Hepes, 0.5
mM EGTA, 150 mM NaCl, pH 7.4, for 24 h. After
dialysis, the resulting proteoliposomes were quickly frozen on liquid
nitrogen, thawed, and sonicated for 5 s in a bath sonicator (Lab
Supplies Co. Inc., Hicksville, NY).
A lipid bilayer, of capacitance >200 picofarads, was
formed by painting a 20 mg/ml solution of a phospholipid mixture
(phosphatidylethanolamine/phosphatidylserine, 50:50) (Avanti Lipids) in n-decane over the aperture in a bilayer chamber. The solution
in the cis compartment contained 300 mM KCl, 10
mM MOPS, 1 mM MgCl
Immunoprecipitation of CFTR from CHO-CFTR
cells revealed a diffuse band of apparent molecular mass of 160 kDa (band C) and a smaller 135 kDa band (bandB) (Fig. 1A). The 160 kDa band represents the mature form
of CFTR (Cheng et al., 1990; Gregory et al., 1990).
It was resistant to digestion with endoglycosidase H but sensitive to N-glycanase (data not shown), consistent with carbohydrate
addition at the Golgi. The smaller bandB represents
the immature or core glycosylated CFTR. CFTR from Sf9 insect cells was
represented by a single band of 130 kDa. This result is not unexpected,
as for many proteins expressed in Sf9 cells, N-linked
oligosaccharides are not processed to complex structures (Altman et
al., 1993).
Several
chromatographic methods were examined for their ability to purify CFTR
from the
Greater than 95% of the
Fig. 3A shows an enrichment of CHO-CFTR purified by
DEAE followed by immunoaffinity chromatography on immobilized mAb
13-1, while Fig. 3B shows an immunoblot of the
same fractions. Pooled DEAE-purified CFTR (lane4)
was estimated to be 20-30% pure by densitometry of the
Coomassie-stained gel. CFTR eluting from the immunoaffinity column (lane5) was found to be approximately 70% pure by
densitometry as shown in Fig. 3C. The overall yield of
CFTR using this two-column procedure was 20-25% (I).
Here we have described the successful reconstitution of
highly purified and functional CFTR from both CHO and Sf9 cells. Stable
CHO cell lines capable of producing 0.3 pg of CFTR/cell and a process
for generating up to 1.7 pg/cell in the baculovirus expression system
have been established. Although CHO cells gave a lower yield of CFTR,
it offered the advantage that it was a continuous and stable cell line
and therefore not subject to varibilities that may arise from batch
infections of Sf9 cells. Furthermore, the glycosylation of CFTR in CHO
cells was more complex and complete than in Sf9 cells. The mature form
of CFTR from CHO cells migrated with an apparent molecular mass of 160
kDa, while that from Sf9 cells was 130 kDa. The 130-kDa molecular size
of Sf9-CFTR is close to the molecular mass of the high mannose
glycosylated form of CHO-CFTR (135 kDa). This result is not unexpected
as for many proteins expressed in Sf9 cells, N-linked
oligosaccharides are not processed to complex structures (Altman et
al., 1993). Insect glycoproteins are first synthesized and
attached to a typical high mannose oligosaccharide with the following
composition [Asn]GlcNAc
The
only report to date of successful purification of functional CFTR used
the baculovirus Sf9 cell system (Bear et al., 1992). However,
because Sf9 cells are unable to effect complete glycosylation of CFTR
and because solubilization of CFTR from Sf9 cells required the use of
strong dissociating conditions such as SDS, we attempted to purify
CHO-CFTR under nondenaturing conditions. The goal was to compare the
relative merits of the purification schemes and the activities of the
purified material from both sources.
We have identified a variety of
nonionic and zwitterionic detergents that were effective at
solubilizing CFTR from the CHO cell membranes (). The most
effective of these solubilizing agents was the lipid
When this immunoaffinity-purified CFTR was
reconstituted into ergosterol containing lipids and fused to a planar
lipid bilayer, a chloride channel activity similar to that of crude
plasma membrane vesicles from CFTR expressing CHO cells was observed
(Tilly et al., 1992). The slope conductance associated with
this partially purified CFTR protein was 10.3 pS (Fig. 10a). This value agrees well with those previously
reported for CHO-CFTR plasma membrane vesicles (6.5 ± 0.3 pS)
recorded in planar lipid bilayers (Tilly et al., 1992) and
CFTR Cl
The immunoaffinity-purified CFTR from CHO cells also
exhibited another Cl
Further purification of CFTR was achieved by incorporating an
ion-exchange chromatography step prior to immunoaffinity chromatography (Fig. 3). The behavior of CFTR on DEAE-Sepharose was the most
consistent of all of the resins tried in this class. The behavior of
CFTR on ion-exchange resins was also shown to be dependent on the type
of detergent used. For example, nonionic, but not zwitterionic
detergents, gave better fractionation. A recurring problem with
fractionation of CFTR based on charge was that CFTR molecules did not
behave as a homogeneously charged population. This resulted in CFTR
eluting from ion-exchange resins over a broad range of salt
concentration, which in turn resulted in less optimal purification and
recovery. This heterogeneity in charge may be due to variations in
glycosylation and phosphorylation of CFTR protein. Thus using a
combination of DEAE and immunoaffinity chromatography, CFTR could be
purified to 60-70% homogeneity with a 25% yield ( Fig. 3and I).
CFTR-mediated chloride currents are activated by 1)
phosphorylation of the R-domain (Cheng et al., 1991; Berger et al., 1991; Tabcharani et al., 1991) and 2)
cytosolic nucleoside triphosphates in the presence of Mg
In summary, we have
described the development of an important prerequisite for protein
replacement therapy for CF, procedures to purify in high yield and
reconstitute functionally active CFTR from two cell sources capable of
producing high levels of CFTR. Purified reconstituted CFTR from insect
and CHO cells exhibited very similar biochemical and
electrophysiological characteristics in terms of chloride channel
activity and ATP binding. The reconstituted system for CFTR described
here may be used as a model for the determination of other biochemical
functions of CFTR such as its ATPase activity.
We thank Dr. R. Gregory and D. Souza for the
baculovirus vectors and A. Pavao, C. Tupper, T. Page, S. Fang, for
technical assistance. We also thank Dr. C. Miller, Brandeis University,
for many helpful discussions and suggestions during this project.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-lysophosphatidylcholine extraction, DEAE ion-exchange,
and immunoaffinity chromatography. Insect CFTR from Sf9 cells was
purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J.,
Ramjeesingh, M. and Riordan, J. R.(1992) Cell 68,
809-818), which included extraction with sodium dodecyl sulfate,
hydroxyapatite, and gel filtration chromatography. Characterization of
the properties of purified CFTR from both cell sources using a variety
of electrophysiological and biochemical assays indicated that they were
very similar. Both the purified CHO-CFTR and Sf9-CFTR when
reconstituted into planar lipid bilayers exhibited a low pS,
chloride-selective ion channel activity that was protein kinase A- and
ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to
interact specifically with the nucleotide photoanalogue
8-N
-[
-
P]ATP with
half-maximal binding at 25 and 50 µM, respectively. These
values compare well with those reported for
8-N
-[
-
P]ATP binding to
CFTR in its native membrane form. Thus CFTR from either insect or CHO
cells can be purified to high homogeneity with retention of many of the
biochemical and electrophysiological characteristics of the protein
associated in its native plasma membrane form. The availability of
these reagents will facilitate further investigation and study of the
structure and function of CFTR and its interactions with cellular
proteins.
(
)a fatal genetic
disorder (Boat et al., 1989) is caused by mutations in the
gene encoding the cystic fibrosis transmembrane conductance regulator
(CFTR). Based on the nucleotide sequence (Riordan et al.,
1989), CFTR was proposed to contain two membrane-spanning domains, a
regulatory or R-domain, and two nucleotide binding domains. Detailed
structure-function studies have now established CFTR as an apically
localized glycoprotein (Cheng et al., 1990; Denning et
al., 1992a) whose Cl
channel activity is
regulated by phosphorylation at the R-domain (Cheng et al.,
1991; Berger et al., 1991; Tabcharani, et al., 1991)
and by cytosolic nucleotide triphosphate binding at both nucleotide
binding domains (Anderson et al., 1991; Anderson and Welsh,
1992; Welsh et al., 1992).
) channel or a regulator of a Cl
channel (Riordan et al., 1989). Early experiments to
dissect these possibilities involved introducing the cDNA encoding
wild-type CFTR into cells from CF patients (Rich et al., 1990;
Drumm et al., 1991) or into cells that did not normally
express CFTR (Anderson et al., 1991; Kartner et al.,
1991; Rich et al., 1991; Bear et al., 1992; Dalemans et al., 1991; Drumm et al., 1991) and showing that
this resulted in the restoration or appearance of a Cl
conductance regulated by cAMP. These results argued that CFTR was
likely to be a phosphorylation-regulated Cl
channel.
Direct and definitive evidence showing that CFTR could act
independently, as a regulated Cl
channel came from
studies by Bear et al.(1992) showing that CFTR highly purified
from Sf9 cells when reconstituted into phospholipid vesicles and fused
to a planar lipid bilayer exhibited cAMP-stimulated Cl
channel activity.
F508)
prevents both the transport and maturation of the glycoprotein to its
apical location (Cheng et al., 1990; Kartner et al.,
1992; Denning et al., 1992a, 1992b). In consequence, these
epithelial cells are unable to regulate normal ion transport, and this,
it is proposed, is the basis for the disease. Several other
CF-associated mutations have been identified (Tsui, 1992), and some
like the
F508 mutation result in synthesis of variants that fail
to localize to the correct cellular location. Others result in the
production of variants that are either unstable or exhibit diminished
Cl
channel activity (for review, see Welsh and
Smith(1993)). Therefore therapies based on reintroduction of a normal
functioning CFTR into these affected cells could ameliorate the
clinical symptoms associated with CF. This may be accomplished either
by gene transfer or by protein replacement therapy.
(
)
Materials
-lysophosphatidylcholine (
-lysoPC), and
ergosterol were from Avanti Polar Lipids (Alabaster, AL).
Expression of Recombinant CFTR
Chinese Hamster Ovary Cells
To generate stable
mammalian cell lines expressing human CFTR, the 4.5-kilobase SalI fragment from the vector pMT-CFTR (Cheng et al.,
1990), which contains the entire human CFTR cDNA, was inserted into the
unique XhoI site of the eukaryotic expression vector
CLH3AXSV2DHFR to generate pDHFR-CFTR (Marshall et al., 1994).
In pDHFR-CFTR, expression of CFTR is controlled by the flanking mouse
metallothionein I promoter. This vector also includes pML sequences and
the gene for dihydrofolate reductase for selection and
methotrexate-mediated amplification of the transfectants. pDHFR-CFTR
was transfected into CHO cells (Urlaub and Chasin, 1980) using the
method of calcium phosphate precipitation (Graham and van der Eb, 1973;
Wigler et al., 1977). CHO transfectants were selected by
growth in media supplemented with methotrexate.
Sf9 Insect Cells
A 4.5-kilobase SpeI-KpnI fragment containing the human CFTR cDNA
together with a Xenopuslaevis -globin leader
sequence at the 5` terminus was cloned between the XbaI and KpnI sites of the transfer vector pVL1392 (Invitrogen) to
generate pBac-CFTR1. Recombinant CFTR-expressing viruses were generated
following co-transfection of Sf9 cells with pBac-CFTR1 and wild-type
AcMNPV DNA essentially as described by the manufacturer (Invitrogen).
Purification of CFTR from CHO Cells
General Considerations
All manipulations
throughout purification were conducted at 0-4 °C in the
presence of protease inhibitors. The starting material was
approximately 300 mg of crude CFTR-containing membranes prepared from 1
10
CHO cells. The highest producing CHO cell line
(designated CU1A), estimated to produce approximately 1
10
CFTR molecules/cell, was used for all purifications.
Estimates of the quantity of CFTR expressed in CHO cells were made
using Western blotting and quantitative densitometry. A standard curve
of pure CFTR (concentration determined by ELISA) was used to determine
the concentration of CFTR in cell lysates from CHO cells. Routinely
CFTR could be purified to 30-40% homogeneity by immunoaffinity
chromatography alone. A more pure preparation (60-70%
homogeneity) of CFTR could be achieved using a combination of
immunoaffinity chromatography and either gel filtration or ion-exchange
chromatography. CFTR was quantified using an ELISA assay (see below).
Membrane Preparation
Cells grown on microcarriers
in 8-liter spinners were harvested by centrifugation at 1,000 g for 5 min. The cell pellets were washed twice with
phosphate-buffered saline, resuspended with approximately 500 ml of
hypotonic lysis buffer (10 mM NaCl, 20 mM Tris-HCl,
pH 7.4, 1 mM EDTA, 2 mM MgCl
, 5 mM dithiothreitol, 10 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A, and 20 µg/ml aprotinin) and allowed to swell on ice
for 30 min. The suspension was then passed through a microfluidizer
(Microfluidics) at 3,000 p.s.i. Unlysed cells and cell debris were
removed by low speed centrifugation (1,000
g), and the
resulting supernatant was recentrifuged at 10,000
g for 25 min. The supernatant was retained and centrifuged at
100,000
g for 1 h to yield a crude membrane pellet
that was resuspended in 150 mM NaCl, 50 mM Tris-HCl,
pH 7.5, 1 mM EDTA, and 10% glycerol.
Alkali Stripping of Crude Cell Membranes and
Solubilization of CFTR
To remove peripherally bound membrane
proteins, the crude membrane fraction was diluted with 10 volumes of 10
mM EDTA, pH 11.0, and kept at 4 °C for 2 min. The
alkali-stripped membranes were collected by pelleting at 100,000
g for 20 min, washed twice with cold
phosphate-buffered saline, and then resuspended at 4 mg/ml in
solubilization buffer (150 mM NaCl, 50 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 10% glycerol, 1.5%
-lysoPC). The
following protease inhibitors were included: 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 mM benzamidine, 5 µg/ml pepstatin A, 5 µg/ml leupeptin, 130
µM bestatin, 50 mM
-macroglobulin, 1 mM Pefabloc, and 250
µg/ml cystatin. The membrane suspension was agitated for 30 min,
and the insoluble membrane fragments were pelleted by centrifugation at
100,000
g for 60 min. The supernatant containing the
solubilized CFTR was decanted and saved, and the pellet was discarded.
Immunoaffinity Chromatography
Monoclonal
antibodies mAb 13-1 (Gregory et al., 1990; Marshall et al., 1994) and mAb 24-1 (Denning et al.,
1992a) were used to prepare immunoaffinity resins. The carbohydrate
moiety of the antibodies was activated with sodium periodate and
coupled to Hydrazide Avidgel (Unisyn Technologies) in 50 mM sodium acetate, pH 5.0. Immunoaffinity chromatography was
performed routinely using either the mAb 13-1 or mAb 24-1
hydrazide resin. CHO membranes containing CFTR were solubilized as
described above, and 25 ml (from approximately 100 mg of membranes) of
the solubilized material was incubated batchwise with 15 ml of resin
for at least 3 h at 4 °C. After collection of the flow-through, the
resin was rinsed with 100 ml of wash buffer (150 mM NaCl, 50
mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% sodium
cholate) to remove nonspecifically bound proteins. Since the precise
epitopes of the monoclonal antibodies have been determined (amino acids
729-736 for MAb 13-1 and 1477-1480 for MAb
24-1, Marshall et al.(1994)), CFTR was eluted from the
resin using elution buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, 0.5% sodium
cholate) containing 5 mg/ml of the appropriate peptide antigen. The
peptide used for elution of CFTR from the 13-1 immunoaffinity
hydrazide resin was SDEPLERRS-NH
, and that used for the
24-1 hydrazide resin was VQDTRL-NH
. Both peptides
were synthesized by QCB (Hopkinton, MA) and were >85% pure by high
performance liquid chromatography. Fractions containing CFTR were
pooled (40-50 ml) and concentrated using a Centricell 20 (30,000
nominal molecular weight limit) to a final concentration of CFTR of 1
mg/ml (500 µl). The immunoaffinity resin was regenerated by washing
with 10 volumes of 0.15 M NaOH, pH 11 (the pH of the stock
NaOH solution (pH 13) was adjusted to 11 using HCl) and stored in 150
mM NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA,
0.02% NaN
at 4 °C. The following protease inhibitors
were added to all buffers immediately before use: 1 mM Pefabloc, 20 µg/ml aprotinin, 10 mM benzamidine, 5
µg/ml pepstatin A, and 5 µg/ml leupeptin.
Gel Filtration Chromatography
Gel filtration
chromatography was performed on a Superdex 200 HR 10/30 column
pre-equilibrated with 150 mM NaCl, 50 mM Tris-HCl, 1
mM EDTA, 10% glycerol, 0.5% sodium cholate, pH 7.5, at a flow
rate of 0.25 ml/min. 200 µl of immunoaffinity-purified CFTR (1
mg/ml) were applied to the column at the same flow rate, and 0.5-ml
fractions were collected. Fractions containing CFTR were detected by
SDS-PAGE followed by silver staining. These fractions were pooled and
concentrated, and CFTR was quantitated by an ELISA.
Ion Exchange Chromatography
A 50-ml column of
DEAE-Sepharose was packed and equilibrated with 10 mM KHPO
, 10% glycerol, 0.05%
-lysoPC, pH
7.5 (conductivity, 3.2 mS/cm). Stripped membranes (100 mg) were
solubilized in the above buffer containing 1.5%
-lysoPC, and the
resulting 100,000
g supernatant was applied to the
column at a flow rate of 2 ml/min. CFTR bound to DEAE-Sepharose under
these conditions. After extensive washing of the resin with
equilibration buffer, a linear gradient of 10-150 mM K
HPO
was applied at a flow rate of 2
ml/min. CFTR eluted from the column at approximately 60-85 mM K
HPO
.
Purification of CFTR from Baculovirus-infected Sf9
Cells
6-cm (30 ml) ceramic hydroxyapatite column was used instead of the
larger 2.6
20 cm (100 ml) Bio-Gel hydroxyapatite column. The
ceramic hydroxyapatite allowed a faster flow rate (0.5 ml/min versus 0.2 ml/min), so the overall time for purification was
decreased from 34 (for hydroxyapatite) to 15 h (for ceramic
hydroxyapatite). Equilibration and elution buffers used on the ceramic
hydroxyapatite column were as described previously by Bear et
al.(1992). The ceramic hydroxyapatite column was equilibrated with
10 mM phosphate buffer, pH 6.4, containing 0.15% SDS and 5
mM dithiothreitol. After washing the resin with equilibration
buffer, a linear gradient of (100-600 mM) of sodium
phosphate containing 0.15% SDS and 5 mM dithiothreitol was
applied. Elution of bound CFTR was achieved with additional washing
with the high phosphate buffer. Additional modifications included
replacing the Superose column in the original protocol with a Superdex
column and performing the chromatography in 0.1% SDS rather than 0.25%
lithium dodecyl sulfate. We found the separation on the Superdex resin
to be more effective than that obtained with the Superose resin. Also
the Superdex resin facilitated purification of larger quantities (mg)
of insect CFTR.
CFTR Protein Detection
ELISA
Photoaffinity Labeling of CFTR
-[
-
P]ATP (azido
ATP, 2-10 Ci/mmol, ICN) in 20 mM Hepes, pH 7.4, 5 mM MgSO
, 100 mM NaCl. Photolabeling was
performed using a handheld UV lamp (model UVG-54 Mineralight from UVP,
Inc.) at a distance of 4.5 cm for 2
30-s intervals. For
competition experiments, the competing nucleotide, ATP, was
pre-incubated with the purified samples of CFTR for 15 min on ice
before the addition of
8-N
-[
-
P]ATP. Reactions
were terminated by the addition of polyacrylamide gel electrophoresis
loading buffer and subjected to SDS-PAGE on a 4-20% gel. The
amount of 8-N
-[
-
P]ATP
incorporated into the CFTR containing band was determined by
densitometric scanning of the Coomassie-stained CFTR band followed by
counting the equivalent area of the gel using a PhosphorImager
(Betascope 603).
SPQ-Halide Efflux Assay
HPO
, 1 mM
CaSO
, 10 mM dextrose, 10 mM Hepes, pH
7.4. After measuring base-line fluorescence for 2 min, the 135 mM NaI solution was replaced with one containing 135 mM
NaNO
, and fluorescence was measured for another 16 min.
Forskolin (20 µM) and isobutylmethylxanthine (100
µM) were added 5 min after the anion substitution to
increase intracellular cAMP. An increase in halide permeability is
reflected by a more rapid increase in SPQ fluorescence. It is the rate
of change rather than the absolute change in signal that is the
important variable in evaluating anion permeability. Differences in
absolute values reflect quantitative differences between groups in SPQ
loading, size of cells, or number of cells studied. The data are
presented as the mean ± S.E. of fluorescence at time t (F
) minus the base-line fluorescence (F
, the average fluorescence measured in
the presence of I
for 2 min prior to ion
substitution) and are representative of results obtained under each
condition.
Reconstitution of CFTR into Phospholipid Vesicles
Planar Lipid Bilayer Studies
, and 2 mM CaCl
, pH 7, and, in the trans compartment,
the concentration of KCl was reduced to 50 mM KCl.
Proteoliposomes (4 µl) were added to the cis compartment
of the chamber followed by stirring to enhance adherence to the
bilayer. Membrane potentials were referenced to the trans compartment, and Cl
current from cis to trans was designated as negative. Single channel activity of
CFTR reconstituted into planar lipid bilayers was acquired via a
current-voltage amplifier (constructed by M. Seng, Department of
Biophysics, University of Alabama, Birmingham) at a sample rate of 1
kHz and recorded on video tape for subsequent analysis. In our studies
of reconstituted protein, we have found that single channels are
typically incorporated into bilayers. Continuous records of single
channel activity, lasting at least 30 s, were analyzed using pCLAMP
software (VI) for determination of current amplitudes.
Generation of CHO and Sf9 Cells Expressing High Levels
of Functional Recombinant CFTR
To facilitate purification of
CFTR, stable CHO cell lines that express large amounts of recombinant
mature CFTR were established. One such clonal cell line, designated
CU1A, produced an average of 1 10
molecules of
CFTR/cell, which equates approximately to 0.33 pg of CFTR/cell. Higher
levels of recombinant CFTR (up to 1.7 pg of CFTR/cell) also were
obtained using the baculovirus expression system in Sf9 cells (Lucknow
and Summers, 1988; Bear et al., 1992). The
baculovirus-infected Sf9 cells also afforded another advantage in that
membranes derived from these cells were enriched for CFTR by
approximately 5-fold when compared with CHO membranes ().
When normalized to total protein content, Sf9-infected cell membranes
on average contained 2.5 times more CFTR than CHO-CFTR membranes. CHO
cells, however, could be grown to 2-3-fold higher cell densities
and were not subject to variability that could arise from batch
infection of Sf9 cells.
Figure 1:
Analysis of CFTR
expressed in CHO and insect Sf9 cells. A, immunoprecipitation
assay of CFTR expressed in CHO and insect Sf9 cells. Lysates of CHO
cells that had been mock transfected (lane 1) or stably
expressing CFTR (CU1A, lane2) and lysates of insect
Sf9 cells (infected with baculovirus expressing CFTR, lane3) or that were uninfected (lane4)
were immunoprecipitated and phosphorylated in vitro with
protein kinase A and [-
P]ATP and analyzed
by SDS-PAGE. The positions of bandsB and C are indicated on the right. B, functional
analysis of CFTR expressed in CHO and insect cells using the SPQ
fluorescence assay. The change in fluorescence of SPQ is shown for 1)
mock transfected CHO cells (n = 10, where n = number of cells), 2) CFTR-CHO cells (CU1A clone; n = 20), 3) Sf9 cells infected with recombinant CFTR
baculovirus (n = 10), and 4) uninfected Sf9 cells (n = 10). NO
was
substituted for I
in the bathing solution at 0 min. 4
min later at the arrow, cells were stimulated with 20
µM forskolin and 100 µM
isobutylmethylxanthine to increase intracellular levels of cAMP. Data
are mean ± S.E. of fluorescence at time t (F)
minus the base-line fluorescence (F, average fluorescence
measured in the presence of I
for 2 min prior to ion
substitution.
The functionality of CFTR produced in these cells
was assessed using the halide-sensitive fluorophore SPQ (Illsley and
Verkman, 1987, Marshall et al., 1994). In this assay, a rapid
increase in fluorescence of SPQ following stimulation with cAMP
agonists is indicative of the presence of functional
phosphorylation-regulated CFTR Cl channel activity. Fig. 1B shows the results from Sf9 cells that had been
infected with a recombinant baculovirus capable of expressing CFTR and
CU1A CHO cells expressing CFTR. Stimulation of these cells with cAMP
agonists (20 µM forskolin and 100 µM
isobutylmethylxanthine) increased SPQ fluorescence indicating an
increased anion permeability. In contrast, parental CHO and noninfected
Sf9 cells displayed no change in SPQ fluorescence upon stimulation with
cAMP agonists (Fig. 1B). Hence expression of CFTR in
both CHO and Sf9 cells results in the appearance of functional
cAMP-activated Cl
channels.
Purification of Recombinant CFTR from CHO Cells
As
an initial step in the purification of CFTR from CHO cells, a crude
membrane fraction was prepared (see ``Experimental
Procedures''). Alkaline extraction of these membranes resulted in
removal of up to 66% of the peripheral proteins while retaining greater
than 80% of the CFTR (data not shown). To extract CFTR from these
stripped membranes, a range of detergents and conditions were screened
for their ability to optimally solubilize CFTR (). Of all
of the detergents screened, -lysoPC was judged to be the most
effective. It was capable of solubilizing 80-90% of CFTR from the
stripped membranes while only solubilizing 50% of the non-CFTR membrane
proteins ().
-lysoPC therefore would appear to be
able to preferentially solubilize CFTR from the stripped membranes.
Another advantage of using
-lysoPC was its compatibility with
ion-exchange and immunoaffinity chromatography. However, since its low
critical micelle concentration (7 µM) (Jones et
al., 1986) made it unsuitable for reconstitution experiments, we
included a detergent-exchange step during immunoaffinity chromatography
in which
-lysoPC was replaced with sodium cholate.
-lysoPC extracts of CHO-CFTR plasma membranes. Gel
filtration, lectin affinity, dye and nucleotide affinity, hydrophobic
interaction, chromatofocusing, and cation-exchange chromatography were
found to be ineffectual either because fractionation of CFTR from its
contaminants was poor or because the recovery of CFTR was low. The
purification scheme that yielded the best results in terms of overall
yield and purity was a combination of DEAE-Sepharose anion-exchange
followed by immunoaffinity chromatography using either mAb 24-1
or 13-1 monoclonal antibodies directed against either the
C-terminal or R-domain, respectively.
-lysoPC-solubilized CFTR bound to DEAE-Sepharose when
chromatographed in 10 mM K
HPO
, pH 7.5. Fig. 2shows that when a linear gradient of 10-150
mM K
HPO
was applied to the resin, CFTR
eluted over a broad range beginning at approximately 60 mM K
HPO
. Generally, by pooling the main
CFTR-containing fractions, 50% of CFTR was recovered that was
approximately 20-30% pure as judged by densitometry of Coomassie
Blue-stained SDS-PAGE gels (data not shown).
Figure 2:
Ion exchange chromatography of CHO-CFTR on
DEAE-Sepharose. The profile of 280 nm absorbance of fractions eluted
from DEAE-Sepharose using a potassium phosphate gradient is shown. A
gradient of phosphate from 10-150 mM
KHPO
was applied at fraction 10 and is
represented by the slopingline. CFTR eluted from the
resin at approximately 60-85 mM K
HPO
corresponding to fractions 40-50. These fractions were
pooled and further purified by immunoaffinity
chromatography.
The CFTR-containing
eluate from the DEAE-Sepharose was further purified by immunoaffinity
chromatography using either immobilized mAb 24-1 or mAb
13-1. Both antibody columns bound greater than 90% of the CFTR
from the DEAE-Sepharose pool. Nonspecific proteins were removed by
washing the resin with 1% sodium cholate in the presence of 150 mM NaCl. This washing step also allowed exchange of the detergent
from -lysoPC to sodium cholate, which was more readily dialyzed
during reconstitution. Nearly all of the bound CFTR was competitively
and specifically eluted from the immunoaffinity resins using synthetic
peptides containing the epitopes of the appropriate monoclonal
antibody. By pooling only the fractions containing the majority of
CFTR, an average recovery of 70% of the bound material was achieved.
Figure 3:
DEAE20/immunoaffinity purification of
CHO-CFTR. A, aliquots containing approximately 1 µg of
total protein were subjected to SDS-PAGE (4-20% acrylamide)
followed by Coomassie Blue staining. Lane1,
molecular weight standards; lane2, alkali-stripped
membrane fraction; lane3, -lysoPC extract of
the same membrane fraction (DEAE-load); lane4, CFTR
containing fractions that were eluted from the DEAE-Sepharose using a
phosphate gradient (also immunoaffinity load); lane5, immunopurified CFTR eluted from the
13-1-hydrazide affinity column. B, immunoblot of CFTR
containing fractions during purification. Lane1,
molecular mass standards; lane2, DEAE load; lane3, pooled fractions eluted from DEAE-Sepharose
(immunoaffinity load); lane4, CFTR containing
fraction eluted from immunoaffinity resin (mAb 13-1) using the
corresponding peptide. Immunoblot was probed with mAb 24-1. C, densitometric scan of purified CHO-CFTR. The purified CFTR
containing-fraction following DEAE and immunoaffinity chromatography
was concentrated and electrophoresed on a SDS-PAGE gel (4-20%)
and stained using Coomassie Blue (Fig. 3A). A densitometric
scan of the Coomassie-stained SDS-PAGE gel is shown. The area of the
gel containing CFTR is indicated. Top and bottom
refer to the orientation of the gel during scanning. Based on this
analysis, the CHO-CFTR was determined to be 70%
pure.
Purification of Recombinant CFTR from Sf9
Cells
Due to the presence of greater amounts of CFTR in the
insect membranes combined with the use of a strong dissociating
detergent (SDS) allowed purification of Sf9-CFTR to greater than 90%
purity. In our hands, the overall yield of purified CFTR was higher
than that previously reported by Bear et al.(1992), which we
attribute to the modifications that we had made to the procedure. This
includes the use of a ceramic hydroxyapatite resin and a Superdex gel
filtration column in the presence of SDS. Using this modified process,
the overall time of purification was decreased, and the yield increased
3-fold to approximately 1.5 mg of CFTR/5 10
Sf9
cells. This represents an overall yield of 15% of CFTR, which was
greater than 90% pure as determined by densitometry. Fig. 4shows
a silver stain and immunoblot of the purified insect CFTR from the
Superdex gel filtration column. The final purified insect CFTR appears
mainly as a diffuse band on SDS-PAGE gels at an apparent molecular mass
of approximately 130-135 kDa (Fig. 4). There is also
evidence of a tighter band of CFTR which runs about 130 kDa (Fig. 4). Insect CFTR is smaller than the CHO-derived CFTR and is
due to the fact that for some proteins expressed in insect cells, only
oligomannosidic structures are added (Altman et al., 1993).
Insect-derived CFTR appeared as a tighter band when solubilized from
the membranes but chromatographed as a diffuse band following
hydroxyapatite chromatography. This is most likely due to the fact that
CFTR desorption from hydroxyapatite occurs in the presence of high
phosphate (600 mM) concentration.
Figure 4:
Analysis
of purified CFTR from insect Sf9 cells. Purified insect CFTR was run on
SDS-PAGE and either silver stained (laneI) or
immunoblotted using mAb 24-1 (lane2). Amino
acid compositional analysis of this fraction determined CFTR to be
greater than 90% pure. The purity of this fraction was also assessed by
Coomassie Blue staining of the protein followed by densitometric
scanning.
Reconstitution of CFTR into Phospholipid
Bilayers
In order to characterize the purified CFTR
biochemically and to assess its chloride channel activity, we
reconstituted the purified material into a lipid environment by
dialysis from cholate (Arion and Racker, 1970; Kagawa and Racker,
1971). Detergent exchange of CHO-CFTR from -lysoPC to cholate was
performed during immunoaffinity chromatography. CHO-CFTR in cholate was
added to sonicated, cholate-solubilized phospholipids and dialyzed for
5 days to form proteoliposomes. Insect CFTR was purified and
reconstituted according to Bear et al.(1992). Fig. 5shows immunoblots of post 100,000
g pellets, which we assume is the fraction of CFTR that was
reconstituted into proteoliposomes. Based on these data, it was
estimated that greater than 90% of the CFTR present was reconstituted
into a lipid environment.
Figure 5:
Immunoblot analysis of reconstituted
insect and CHO-CFTR. Following detergent dialysis of purified insect (A) or purified CHO (B) CFTR in the presence of
lipids, the resulting proteoliposomes were recovered by
ultracentrifugation at 100,000 g, electrophoresed on
SDS-PAGE gels, and then immunoblotted using mAb 24-1. A, lane1, post 100,000
g pellet
containing insect CFTR proteoliposomes; lane2,
supernatant from 100,000
g centrifugation. B, lane3, CHO-CFTR in detergent and lipids prior to
dialysis; lane4, post 100,000
g supernatant of CHO-CFTR and lipids after dialysis; lane5, post 100,000
g pellet of
proteoliposomes containing CFTR.
Experiments to determine the orientation
of the reconstituted CFTR were performed using
8-N-[
-
P]ATP. In an
intact proteoliposome, this photolabel should only label the nucleotide
binding domains of CFTR that are exposed on the vesicle surface.
Proteoliposomes containing CFTR were labeled with
8-N
-[
-
P]ATP in the
presence and absence of detergent. The percentage increase in total
8-N
-[
-
P]ATP labeling
of CFTR in the presence of detergent (i.e. both inside-out and
right side-out molecules will label under these conditions) was used to
determine the percentage of total CFTR that was reconstituted
inside-out. Using this method, it was determined that 60% of the
reconstituted CFTR had the R-domain and nucleotide binding domains
extravesicular (data not shown).
Interaction of Purified CFTR with Nucleotides
We
also employed photolabeling of CFTR with
8-N-[
-
P]ATP to assess
whether the purified CFTR in detergent and in reconstituted vesicles
retained the same binding characteristics for
8-N
-[
-
P]ATP as that
reported for 8-N
-ATP binding to insect CFTR in its
native membrane form (Travis et al., 1993). Retention of the
ability of the highly purified CFTR to bind ATP would suggest that the
integrity of the nucleotide binding sites was maintained during
purification. Fig. 6shows that CFTR in crude CHO membranes was
specifically photolabeled by
8-N
-[
-
P]ATP.
Photolabeled CFTR was immunoprecipitated from CHO-CFTR membranes that
had been prelabeled with
8-N
-[
-
P]ATP. Labeling
was virtually totally inhibited if the membranes were preincubated with
cold ATP prior to
8-N
-[
-
P]ATP labeling
and immunoprecipitation (Fig. 6). Thus CFTR can be labeled in its
native membrane form with 8-N
-ATP. Further
experiments were performed to determine the characteristics of this
binding.
Figure 6:
Immunoprecipitation and photolabeling of
CFTR in CHO cell membranes. CHO cell membranes (100 µg) were
photolabeled with
8-N-[
-
P]ATP (100
µM) in the presence or absence of ATP (2 mM).
CFTR was then immunoprecipitated from the membranes using mAb
24-1 and analyzed on a SDS-PAGE (4-20%) gel. Lane1, molecular weight standards; lane2,
immunoprecipitate of CHO-CFTR membranes photolabeled with
8-N
-[
-
P]ATP; lane3, immunoprecipitate of CHO-CFTR membranes photolabeled
with 8-N
-[
-
P]ATP in
the presence of excess ATP (2 mM).
To test for saturability of 8-N-ATP
binding to CFTR and to obtain an estimate of the binding affinity,
photolabeling of CFTR over a range of 8-N
-ATP
concentrations in the presence or absence of excess cold ATP (10
mM) was determined. Reconstituted, purified CHO or insect CFTR
was used for these experiments. Photolabeling of reconstituted insect
CFTR saturated at 100 µM 8-N
-ATP with
half-maximal binding at 40-50 µM 8-N
-ATP (Fig. 7). Similarly with
reconstituted CHO-CFTR, photolabeling saturated at approximately 50
µM 8-N
-ATP with half-maximal binding
at approximately 20 µM ( Fig. 7and ).
That these values compare very well with those obtained for Sf9-CFTR
from crude membranes (saturation at 60 µM and half-maximal
labeling at 10 µM, Travis et al.(1993)) suggests
that the integrity and function of the nucleotide binding domains of
the highly purified CFTR obtained here were intact and preserved.
Figure 7:
Azido ATP labeling of purified
reconstituted CFTR. Graphical representation of a dose-response curve
for the binding of
8-N-[
-
P]ATP to both
reconstituted insect and CHO-CFTR is shown. Purified, reconstituted
CFTR (10 µg) was photolabeled with increasing concentrations of
8-N
-[
-
P]ATP
(10-200 mM) in the presence and absence of excess cold
ATP (10 mM). Samples were analyzed on a SDS-PAGE gel, and
photolabeled areas were quantitated using a PhosphorImager. For
Sf9-CFTR (
) proteoliposomes, photolabeling saturated at 100
µM
8-N
-[
-
P]ATP with
half-maximal binding at 45 µM
8-N
-[
-
P]ATP. For
reconstituted CHO-CFTR (
) photolabeling saturated at 50
µM 8-N
-ATP with half-maximal binding
at approximately 20 µM
8-N
-[
-
P]ATP.
ATP Inhibition of 8-Azido-ATP Labeling
As shown
above, high concentrations of ATP were able to inhibit azido-ATP
photolabeling of Sf9-CFTR either when reconstituted in its native
membrane or in artificial proteoliposomes ( Fig. 6and Fig. 7). The nature of this inhibition was further studied by
examining the effects of
8-N-[
-
P]ATP binding to
purified CFTR in the presence of increasing concentrations of cold ATP. Fig. 8shows that half-maximal inhibition of
8-N
-[
-
P]ATP labeling
to either purified reconstituted CHO or insect CFTR occurred at around
1 mM ATP (). This value is consistent with those
reported for
8-N
-[
-
P]ATP binding to
CFTR in its native membrane (Travis et al., 1993) and from
results of functional studies with CFTR in cell-free membrane patches
(Anderson et al., 1991). In these studies, half-maximal
Cl
channel activity required 270 µM ATP,
while in the sweat duct cells (Quinton and Reddy, 1992) or T84 cells
(Bell and Quinton, 1993) concentrations of greater than 1 mM ATP were necessary for chloride channel function. In conclusion,
these results suggest that the binding sites for
8-N
-ATP on the purified CFTRs were not grossly
affected by the processes used for their purification.
Figure 8:
Inhibition of azido labeling of CFTR by
ATP. Purified and reconstituted CFTR (10 µg) was photolabeled with
8-N-[
-
P]ATP in the
presence of increasing concentrations of ATP (0-10 mM).
The ATP inhibition curves for
8-N
-[
-
P]ATP binding to
reconstituted (
) Sf9-CFTR or (
) CHO-CFTR are shown. All
values are expressed as a percent of control binding of
8-N
-[
-
P]ATP to
reconstituted CFTR.
Chloride Channel Activity of Purified CFTR
We had
previously shown in a planar lipid bilayer assay that crude membrane
vesicle preparations from our CHO-CFTR cells exhibited a low
conductance (6.5 pS) channel that was dependent on the presence of
protein kinase A and ATP (Tilly et al., 1992). To assess
whether the protocols described here for purification of CFTR affected
this chloride channel activity, proteoliposomes containing the purified
CFTR were examined using this same assay. Initial experiments were
performed using immunoaffinity-purified CFTR that was approximately 40%
pure (Fig. 9). As in the studies of insect CFTR protein purified
from Sf9 cells (Bear et al., 1992), we used the nystatin
fusion method described by Woodbury and Miller(1990) to verify fusion
between liposomes containing CFTR and the planar lipid bilayer.
Nystatin forms a nonselective channel in the presence of ergosterol,
and liposomal fusion events are detected as transient conductance
spikes. We found that partially purified CHO-CFTR was capable of
protein kinase A-stimulated channel activity. The predominant
conductance, observed in four of seven trials is shown in Fig. 10a. In the presence of a KCl gradient between the cis and trans compartments of the bilayer
compartments (300 and 50 mM, respectively) the slope of the
current-voltage relation for this channel was 10.3 pS, and the reversal
potential was 20 mV, consistent with the channel being anion selective.
The conductive and regulatory properties of the CHO-CFTR were similar
to those observed for highly purified Sf9-CFTR. In the present studies,
the chloride channel activity of Sf9-CFTR was protein kinase
A-dependent and exhibited a unitary conductance of 10-11 pS (Fig. 10b) as in the original report by Bear et
al.(1992).
Figure 9:
Immunoaffinity purification of CHO-CFTR.
CHO-CFTR was purified using immobilized mAb 13-1 as an
immunoaffinity resin. The peptide eluted fraction was analyzed on a
SDS-PAGE gel, Coomassie-stained, and purity assessed by densitometric
analysis. Immunoaffinity purified CFTR was determined to be
40-50% pure. Lane1, molecular weight
standards; lane2, peptide eluted CFTR-containing
fraction.
Figure 10:
CFTR partially purified from CHO
membranes exhibits characteristic chloride channel activity. a, this record shows a typical single channel recording from
CHO-CFTR liposomes after fusion with planar lipid bilayer in the
presence of MgATP (1 mM) and catalytic subunit of protein
kinase A (200 nM). The channel was exposed to asymmetrical KCl
solutions with 300 mM KCl in the cis and 50 mM in the trans compartment (ground). The applied potential
was -40 mV, and the arrow indicates the current level
for the closed state of the channel. b, channel activity
exhibited by CHO-CFTR is similar to that of highly purified Sf9 CFTR as
shown in this record. c, the slope conductance of CHO-CFTR
channel activity shown in a was 10 pS, as determined from this
current-voltage relationship.
Occasionally other channels were evident following
bilayer fusion of proteoliposomes containing CHO-CFTR. As seen in Fig. 11an 80-pS chloride selective channel could infrequently be
observed along with the CFTR conductance. i.e. three of seven
trials. A similar, 80-pS chloride channel has been detected in lipid
bilayer studies of CHO plasma membranes.(
)However, unlike the CFTR channel, this larger
conductance did not require phosphorylation by protein kinase A for
activity. Purified and reconstituted CFTR from Sf9 cells exhibited only
a low unitary chloride conductance of 11-13 pS that was dependent
on protein kinase A and ATP, characteristics of CFTR. As the
Sf9-derived CFTR was of higher purity, it is likely that the larger
conductance channel observed in the CHO-derived preparations was due to
a contaminating channel that had been co-purified with CFTR.
Figure 11:
An 80-pS chloride channel is a
contaminant of preparation. Infrequently, a large, 80-pS chloride
selective channel is observed along with a 10-pS channel following
fusion of liposomes containing partially purified
CHO-CFTR.
-Man
-Glc
(Hsieh and Robbins, 1984; Jarvis and Summers, 1989). Normally, in
mammalian cells, further processing of N-linked glycans leads
to the addition of a trimannosyl core
([Asn]GlcNAc
-Man
) to the protein. In
insect cells, the trimannosyl core represents the fully processed
oligosaccharide (Miller, 1988). In CHO cells, addition of complex
carbohydrates to the mannose core occurs in the Golgi resulting in a
protein of higher molecular size. The presence of complex carbohydrate
on CHO-CFTR is more representative of the type of glycosylation that
normally occurs on CFTR in airway epithelial cells. Finally,
solubilization of CFTR from CHO membranes could be effected with
greater ease and milder conditions than from Sf9 cell membranes.
-lysoPC,
which was able to selectively solubilize 80% of CFTR but only 50% of
the total cell protein. Several purification strategies were developed
for the solubilized CFTR. A rapid, one-step immunoaffinity purification
procedure using specific monoclonal antibodies against CFTR facilitated
purification of CFTR to 40% with 50% recovery (Fig. 9). For
immunoaffinity chromatography, the choice of detergent for membrane
protein solubilization was restricted to the use of relatively
nondenaturing detergents. In this study, we found that the antigenic
determinants for monoclonal antibodies 13-1 (anti-R-domain) and
24-1 (anti-C-terminal) were well preserved when CFTR was
solubilized with
-lysoPC and a range of other detergents (listed
in ). Elution of proteins from immunoaffinity columns
normally requires harsh conditions such as low or high pH or the use of
chaotropic agents that may denature the proteins. In this case, CFTR
was competitively eluted from the immunoaffinity resin using the
relevant peptide epitope.
channels in excised membrane patches
(8-10 pS) (Bear et al., 1992; Berger et al.,
1991; Kartner et al., 1991; Tabcharani et al., 1991).
This single channel conductance of CHO-CFTR (10.3 pS) is also very
similar to that observed with highly purified Sf9-CFTR. In this study,
the chloride channel activity of Sf9-CFTR was protein kinase
A-dependent with a unitary conductance of 10-11 pS (Fig. 10b) in agreement with the original report by Bear et al.(1992). Thus the Cl
channel activity
of CFTR that had been extracted from CHO cell membranes with
-lysoPC, purified using immunoaffinity chromatography, and
reconstituted into proteolipisomes, was similar to that found in native
membranes.
channel with a conductance of
approximately 80 pS (Fig. 11). This channel has been observed
previously in bilayer studies of CHO cell membranes expressing
CFTR.
To further purify the CHO-CFTR and eliminate this
intermediate conductance channel, we evaluated gel filtration and
ion-exchange chromatography. Gel filtration chromatography on Superdex
200 resin was judged not to be very effective. This is contrary to what
was reported by Bear et al.(1992) for the purification of
insect CFTR. However, a strong dissociating detergent SDS/lithium
dodecyl sulfate was used by Bear et al.(1992) in their
chromatography. Since CFTR is predicted to have several hydrophobic
domains (Riordan et al., 1989) it is likely that it may have
hydrophobic interactions with other membrane proteins, thereby making
it difficult to isolate unless in the presence of a dissociating
detergent such as SDS. In our attempt to purify CFTR under
nondenaturing conditions, we had used less denaturing detergents such
as sodium cholate. Under these conditions, we found it difficult to
disrupt these protein-protein associations and see any improvement on
purification with gel filtration chromatography (data not shown).
(Anderson et al., 1991). The interaction of MgATP with
CFTR can be altered by mutations in either nucleotide binding domain,
suggesting that ATP interacts directly with both nucleotide binding
domains in regulating the channel (Anderson and Welsh, 1992). As ATP is
required both as a channel agonist and a phosphoryl donor for protein
kinase A, it is difficult to assess whether ATP hydrolysis is coupled
to CFTR activity. In this study, we looked at the interaction of
8-N
-ATP with our purified insect and CHO-CFTR. The
characteristics of binding of this photoanalogue has been reported for
insect CFTR in its native membrane (Travis et al., 1993). We
show that CFTR (both insect and CHO) could be purified and
reconstituted into artificial lipids and still retain the same
characteristics for binding 8-N
-ATP () as crude CFTR-containing membranes. This suggests that
the integrity of the binding site for ATP had been preserved during the
purification of CFTR and that the purified reconstituted protein may be
used as a good system to determine if ATP hydrolysis is involved with
CFTR gating. Reports from Anderson et al.(1991) propose that
channel opening required ATP hydrolysis. This has also been suggested
recently by Gunderson and Kopito(1994), while other investigators
(Quinton and Reddy, 1992; Bell and Quinton, 1993) have proposed a
nonhydrolytic role for ATP in CFTR gating.
Table: Comparison of production of recombinant CHO and
insect CFTR
Table: Detergent solubilization of CFTR
Table: DEAE-Sepharose/immunoaffinity purification of
CHO-CFTR
Table: Characteristics of binding of azido-ATP to
purified CFTR
-lysoPC,
-lysophosphatidylcholine; mAb,
monoclonal anti-body; PAGE, polyacrylamide gel electrophoresis; CAPS,
3-(cyclohexylamino)propanesulfonic acid; MOPS,
4-morpholinepropanesulfonic acid; SPQ,
6-methoxy-N-(3-sulfopropyl)quinolinium; S, siemen(s).
Namee, M. G. (1986) in Solubilization and Reconstitution of Membrane Proteins in Biological Membranes: A Practical Approach (Findlay, J., ed) IRL Press
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.