Note: Descriptions are shown in the official language in which they were submitted.
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1
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
Protein
This invention relates to purified and functionally
reconstituted preparations of Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) and to pharmaceutical
compositions and methods of use employing these
preparations.
The discovery of the gene which is mutated in patients
with cystic fibrosis (CF) and the principal disease-causing
mutation (Rommens et al., 1989; Riordan et al., 1989; Kerem
et al., 1989) has given rise to the possibility of the
development of molecular therapies. These can be
considered in at least three broad categories: A.) The
creation or identification of drugs to appropriately modify
CFTR function or biosynthesis; B.) gene therapy by the
delivery of the CFTR DNA sequence in an appropriate vector
to affected epithelial cells; C.) protein replacement
therapy in which the CFTR protein in an appropriate vehicle
is delivered to the same cells.
The steps to be accomplished for the effective
application of the third strategy 1) the production of
large quantities of functional CFTR protein; 2) the
solubilization and purification of the CFTR protein; 3) the
reconstitution of the homogeneous purified protein into a
lipid environment in which it can function; 4)
demonstration that the purified and reconstituted CFTR
molecule has the same functional properties as it had in
the epithelial cells to which it is native; 5) fusion of
proteoliposomes containing functional purified CFTR with
the apical surfaces of CF epithelial cells expressing non-
functional mutant CFTR or no CFTR at all in order to
restore regulated chloride channel activity.
The original CFTR cDNAs which we had isolated and
cloned (Riordan et al., 1989) and deposited with ATCC have
been used for expression of CFTR in a number of different
heterologous mammalian cell systems (Tabcharani et al.,
1991; Anderson et al., 1991a; Cheng et al, 1990; Dalemans
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et al., 1991). However, because of limitations on the
amount of CFTR which can be synthesized in human and other
mammalian cells (Cheng et al., 1990; Gregory et al., 1991),
it was necessary to utilize an alternative system to
generate adequate amounts for purification. We employed
the baculovirus expression vector system (BEVS; Lucknow and
Summers, 1988) to produce large quantities of functional
human CFTR in insect Sf9 cells (Kartner et al., 1991).
More recently, others have produced CFTR protein in the
milk of transgenic mice (DiTullio et al., 1992) as another
potential means of producing sufficient protein for
purification. However, in that work no evidence of
functionality was demonstrated, nor were any attempts at
purification made.
The present invention involves the fulfilment of steps
2.), 3.) and 4.) resulting in the production of highly
purified CFTR protein as judged by stringent criteria of
homogeneity. The purified protein is further demonstrated
to exhibit the same functional properties of a regulated
chloride ion channel as it does in its native location in
vivo. In addition, as expected, structural features
including N-terminal amino acid sequence (6 residues),
overall amino acid composition and isoelectric point are
identical to those predicted from the translated DNA
sequence of the coding region of the cloned CFTR gene. The
only feature of the protein produced in the insect cell
expression system which differs from that produced in human
epithelial cells in the type of carbohydrate added when the
protein is glycosylated during synthesis. However, we have
already demonstrated that this difference is without
influence on the function of the glycoprotein (Kartner et
al., 1991). The glycosylation of the protein in any other
of the alternate expression systems which may be used such
as milk of transgenic animals (DiTullio et al., 1992) will
also differ from that in the human lung which will be the
principal site of delivery for therapeutic purposes.
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The invention also teaches that the proteoliposomes of
the type known to be capable of fusing with the membranes
of cells, can be fused to planar lipid bilayers in which
the generation of electrical currents carried by chloride
ions through the CFTR channel can be measured.
In accordance with a further aspect of the invention,
there is provided a means of replacement of defective CFTR
in the epithelial cells from CF patients by delivery to
them of the purified and reconstituted recombinant CFTR
protein.
In addition to its direct use in protein replacement
therapy, the purified CFTR of the invention provides for
the development of alternative therapeutic strategies, for
example the development of rationally designed drugs based
on features of the molecule's structure which can be
determined from the purified preparation.
Background of the Invention
The cloning of the gene mutated in patients with cystic
fibrosis (CF) has made possible interpretation of the
deduced primary structure of the gene product, CFTR. In
the context of what was known of an epithelial C1-
permeability defect in CF, this lead to the original
suggestion that the gene coded for either a C1- channel
itself or a regulator of a separate C1' channel (Riordan et
al, 1989). The introduction of expressible CFTR cDNAs into
cells bearing CF-causing mutations in the gene (Rich et al,
1990; Drumm et al, 1990), or into cells in which CFTR is
not normally expressed (Anderson et al, 1991a; Kartner et
al, 1991; Rich et al, 1991; Bear et al, 1991; Dalemans et
al, 1991; Drumm et al, 1991) resulted in the appearance of
a Cl' conductance regulated by cyclic AMP and similar to
that seen in several normal epithelial cell types (Gray et
al, 1989; Tabcharani et al, 1990). A low conductance ohmic
Cl' channel activated by protein kinase A (PKA) - catalysed
phosphorylation and inactivated by dephosphorylation was
shown to underlie this conductance pathway (Tabcharani et
al, 1991; Berger et al, 1991). Although these findings
CA 02061579 2002-02-06
4
cannot distinguish between the CFTR protein
constituting the conductance pathway itself, or its
being a phosphorylation-activated regulator, changes
in ion selectivity on mutation of amino acids with
charged side chains in the proposed transmembrane
sequences (viz. K95 in TM1 and K335 in TM6; Anderson
et al, 1991b) tend to support the former possibility.
Consistent with its proposed role as an ion
channel, CFTR is a relatively non-abundant protein in
the epithelial tissues in which it is endogenously
expressed. We know of no tissue which provides an
adequate source for purification. Similarly, it has
not yet been possible to establish mammalian cell
lines in which a very high level of heterologous
expression of CFTR occurs (Cheng et al, 1990). This is
believed to be at least partially due to a rather
stringent control of CFTR biosynthesis which limits
the amount of wild type protein which accumulates in
cells (Gregory et al, 1991). This quality control is
apparently more strictly enforced in the case of some
mutant forms of CFTR, including the product of the
most common mutation (F508), in which little or no
mature protein is detectable and only small amounts of
immature precursor is present, apparently in the
endoplasmic reticulum (Cheng et al, 1990).
Until the work of the present inventors, no one
had succeeded in isolating CFTR and purifying it to
substantial homogeneity.
In accordance with an aspect of the present
invention there is provided a substantially
homogeneous protein having cystic fibrosis
CA 02061579 2007-06-01
4a
transmembrane conductance regulator activity and characterised
by migration as a single band on both one- and two-dimensional
gel electrophoresis.
In accordance with another aspect of the present invention
there is provided a method for purifying a recombinant
hydrophobic membrane protein comprising the steps of:
(a) providing a sample of cells containing a membrane-
associated protein to be purified;
(b) disrupting the cells and pelleting a particulate fraction
thereof;
(c) contacting the particulate fraction with a dilute alkali
solution for an effective period of time at an effective
temperature to extract unwanted constituents followed by
repelleting the particulate fraction;
(d) dissolving the particulate fraction in a buffered
denaturing detergent solution containing a thiol reducing
agent;
(e) subjecting the solution obtained by step (d) to
hydroxyapatite chromatography to provide a partially purified
protein;
(f) subjecting the partially purified protein obtained by step
(e) to molecular sieve chromatography to provide a
substantially homogeneous protein; and
(g) renaturing the protein obtained by step (f) by
competitively removing the denaturing detergent from
association with the protein by exposure to excess non-
denaturing detergent and removing the non-denaturing detergent
and the denaturing detergent by dialysis in the presence of
phospholipid whereby the protein is incorporated into
CA 02061579 2003-10-23
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phospholipid vesicles to provide a functional protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Enrichment of CFTR during major steps
of purification. Aliquots containing approximately 1
ug of protein were subjected to SDS-PAGE (6%
acrylamide in A and B and 4-15% acrylamide in C).
Panels A and C show results of silver staining and B
is an inununoblot probed with monoclonal antibody
M3A7. In A and B, lane 1 is the initial crude
particulate fraction; lane 2, the same fraction after
alkali extraction of peripheral membrane proteins;
lane 3, the highly enriched peak 'F' from
hydroxyapatite; lane 4, the CFTR-containing fraction
of the
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final Superose 6 step. In panel C only the final gel
filtration purified fraction was run.
Figure 2. Major purification of CFTR by hydroxyapatite
chromatography. The upper panel shows the elution profile
5 with phosphate gradient indicated and the lower panel shows
silver staining protein bands after SDS-PAGE of fractions
as indicated. Lane F containing most of the CFTR clearly
corresponds to lane 3 in panels A and B of Figure 1.
Figure 3. Gel filtration chromatography on Superose 6
of peak F from Figure 2. Fractions constituting peak 1
contain highly purified CFTR as indicated in lane 4 of
Figure I A and B, table II and N-terminal sequence
analysis.
Figure 4. 2-D gel electrophoresis of purified CFTR.
For isoelectric focusing 2% ampholytes ranging from pI 3.5
to 10 and 7% acrylamide was used. Electrophoresis was as
with 1-D gels as was silver staining (A) and immunoblotting
(B).
Figure S. Electronmicroscopy of negative stained
proteoliposomes containing CFTR.
Vesicles on carbon formvar coated grids were stained with
2% uranyl acetate. Scale is 0.1 m.
Figure 6. Low Conductance C1' Channel Associated with
CFTR Expression in Sf9 Cells.
(A) This panel shows PKA-activated single channel current
tracings at various pipette potentials in excised patches.
The pipette and the bath contained symmetrical salt
solutions (NaCl = 140 mM). In addition, PKA (200 nM) and
ATP (1 mM) were added to the bath to stimulate the
appearance of this channel. (B) Currents tracings from
PKA-stimulated channels in excised membrane patch from
CFTR-expressing Sf9 cell. In this case, channels were
studied in the presence of 300 mM NaCl in the bath and 50
mM NaCl in the pipette. (C) Current-voltage relationships
are shown for PKA-stimulated channel in symmetrical NaCl
solutions (bath and pipette =140 mM) (o) (n=8) and
asymetrical NaCl solutions (bath=300 and pipette =50 mM
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NaCl) (=) (n=9). Means and SD have been shown. Arrows
indicate the closed conductance state.
Figure 7. Purified CFTR functions as a phosphorylation
activated ion channel in lipid bilayers
(a) The upper trace shows four nystatin spikes ( o ) which
fail to lead to single channel activity. Two nystatin
spikes are long lasting and two short lasting, the
differences possibly reflecting stochastic variation in the
number of nystatin units per liposome. Scale bars indicate
1 pA vertically and 5 sec horizontally. The lower trace
shows a short lasting nystatin spike which is followed by
the appearance of single chloride channel activity. PKA
and Mg-ATP are present in both the cis and trans
compartments. The scale bars indicate 1 pA vertically and
2 sec horizontally. Holding potential is -25 mV.
(b) Addition of liposomes which do not contain purified
CFTR to bilayer chamber with PKA (200 nM) and Mg2ATP (1 mM)
fails to cause appearance of unitary current steps. (c)
Addition of liposomes containing CFTR with no added PKA and
Mg2ATP fails to evoke single channel activity. (d) Single
channel activity is apparent only in those experiments in
which CFTR-containing liposomes are added to the bilayer
chamber with PKA and Mg2ATP. The applied potential was -25
mV in this experiment.
Figure S. Comparison of current-voltage relationship
of PKA-stimulated purified CFTR and C1" channel in T84 cells
and CFTR-expressing CHO cells.
(A) Current traces are shown for purified CFTR protein in
lipid bilayer at various potentials. The cis compartment
of bilayer chamber contained 300 mM KCI, PKA (200 nM) and
Mg-ATP (1 mM). The trans compartment contained 50 mM KCI
+ PKA and Mg-ATP. (B) I-V relationships are shown for
conductance of PKA-activated purified protein with 300 mM
KCI in the cis compartment and 50 mM KCI (e) (n=6) or 10 mM
KCI (o) (n=4) in the trans compartment. (C) The upper
panel shows two 11 pS channels opening sequentially in
stepwise manner. The lower panel shows a larger
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conductance observed in the same recording which
corresponds to twice the conductance of the more prevalent
smaller conductance and may represent cooperative gating of
two 11 pS channels. Holding potential was -45 mV. (D)
I-V relationships for PKA-activated purified CFTR (0)
(n=4), PKA-activated chloride channel in T84 (e) (n=4) and
PKA-activated CFTR-expressing CHO (=) (n=4) membranes
studied in planar lipid bilayers (cis:trans=300:10 mM KC1) .
Description of the Invention
The inventors showed previously (Kartner et al, 1991)
that large amounts of functional CFTR can be generated in
Sf9 insect cells using the baculovirus expression vector
system (BEVS; Luckow and Summers, 1988), thereby providing
the starting point for purification. Suspension cultures
of these cells have been used to obtain relatively large
quantities of a crude CFTR-containing particulate fraction
as starting material for purification. Cold alkaline
extraction (Steck and Yu, 1973) of the particulate fraction
resulted in removal of approximately 2/3 of the total
protein while retaining CFTR (Table I; Figure 1 A and B).
At this stage a CFTR band was clearly visible by protein
staining following SDS-PAGE (Figure 1A). Our strategy for
solubilization and further fractionation employed the
strong dissociating conditions of an ionic detergent for
two principal reasons. First, systematic testing of the
effectiveness of a range of non-ionic and ionic detergents
to solubilize CFTR in membranes of T84 epithelial cells,
CHO cells or Sf9 cells expressing the protein showed that
only the stronger ionic ones were effective. Second,
because our major aim was to determine whether CFTR could
function as a regulated Cl' channel we wished to minimize
the possibility of copurification of any proteins which
might associate with CFTR and contribute to the function of
the final purified material.
Conventional column chromatography techniques
compatible with the presence of sodium dodecyl sulfate
(SDS) (at 0.15 to 0.25 %) were then tested for their
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ability to separate CFTR from other proteins. Among the
methods attempted, adsorption to hydroxyapatite proved to
be by far the most effective (Figure 2). CFTR interacted
with the matrix more strongly than nearly all other
5;proteins under the conditions employed and was eluted only
after the phosphate gradient had reached its maximum
concentration of 600 mM. A purification of at least one
hundred fold was achieved in this step (Table 1). A major
contaminating protein of approximately 30 kd, and very high
molecular weight material running at the origin of a 6%
acrylamide gel, was also present in this fraction. Minor
amounts of faintly silver staining material at molecular
weights both lower and higher than the principal CFTR band
were also detectable; the immunoblot of this lane indicates
that at least some of these are degraded and aggregated
forms of CFTR, respectively. CFTR could be separated from
the remaining contaminants by gel filtration on Superose 6
(Figure 3). The protein eluted in a well-resolved included
peak corresponding to 28% of the Superose gel volume. From
one liter of cells (5 x 109) approximately 0.5 mg of CFTR
protein was obtained in this peak.
Characterization of purified CPTR
The effectiveness of the major purification steps is
summarized in Figure IA and B. The presence in the final
product assessed on 6% SDS-PAGE gels of a single silver
staining band reactive with monoclonal antibodies to CFTR
indicates that it is not contaminated with other proteins
larger than the cutoff molecular weight of the gel. To
determine if still smaller proteins might be present a 4 -
15% gradient gel was heavily loaded and stained with silver
(Figure IC). No other bands were detectible.
As a more stringent assessment of homogeneity, two
dimensional gel electrophoresis (Dottin et al, 1979) was
performed and analysed by silver staining and
immunoblotting (Figure 4). As in the 1-D gels no
contaminating proteins were detected. The major
isoelectric form of CFTR migrated at a position
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9
corresponding to a pI of approximately 9. This agrees well
with the value of 8.98 calculated from the CFTR amino acid
composition. Since only core, mannose-containing N-linked
oligosaccharides are added to proteins in Sf9 cells
(Vialand et al, 1990) carbohydrate would not be expected to
influence the pl. The small immunoreactive spots may
represent alternate isoelectric forms of CFTR.
The final purified protein was subjected to both
N-terminal sequence determination and quantitative amino
acid compositional analysis. The sequence of the
N-terminal 6 residues agreed with that predicted from the
DNA sequence for CFTR and there was excellent agreement
between the amounts of each residue in the overall
composition. The amino acid composition compared with that
deduced from the sequence is shown in table II.
CFTR Reconstitution into phospholipid vesicles
In order to be able to determine the functional capacity of
purified CFTR it was necessary to transfer the detergent
solubilized protein back into a lipid environment. This
was done by a dialysis protocol analogous to that employed
for renaturation of bacteriorhodopsin (London and Khorana,
1982; Braiman et al, 1987) and some other transport
proteins. Essentially, pure CFTR in 0.25% lithium dodecyl
sulfate (LiDS) was mixed with a sonicated phospholipid
mixture (PE:PS:PC at 5:2: 1) containing 1 % cholate and
dialysed extensively to form proteoliposomes. Following
dispersion by sonication, and concentration, these
proteoliposomes were fused with liposomes of the same
phospholipid composition but also containing ergosterol and
nystatin to promote and enable detection of subsequent
fusion to planar lipid bilayers (see below). This
modification to enable the nystatin-induced liposome fusion
was taken from Woodbury and Miller (1990). Following
elution in the void volume of a Superose 12 gel filtration
column, all of the CFTR employed in the reconstitution
could be accounted for in immunoblots of the
proteoliposomes.
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Negative staining indicated a uniform population of
CFTR containing proteoliposomes, about half of which have
a diameter of 40-60 nm with the other half at approximately
15-20 nm (Figure 5). Some fused larger vesicles were also
5 present. The 50 nm vesicles would have a surface area of
7.9 x 105 A2. Using a value of 50 KZ for the average area
occupied by a phospholipid molecule (Levitzki, 1985) there
should be 1.6 x 104 phospholipid molecules per vesicle.
Since we used 2 mg of phospholipid or 1.5 x 1018 molecules
10 approximately 1014 vesicles will have formed. These had
incorporated 6.38 g (based on quantitative amino acid
analysis) corresponding to 3.8 x 10'11 moles or 2.3 x 1013
molecules of CFTR. Therefore, not more than one
(approximately 0.23) CFTR had incorporated per 50 nm
vesicle. The number of these vesicles which were
subsequently fused to a black lipid film could then be
monitored by the nystatin mediated conductance spikes (see
below).
Effect of CyTR-aontaining proteoliposomes on a planar
bilayer
A cyclic AMP-activated, low-conductance chloride
channel has been described in CFTR expressing Sf9 cells
(Kartner et al., 1991). In order to compare the conductive
properties of purified CFTR with that of the
CFTR-associated channel in native membranes, it was first
necessary to characterize this channel in Sf9 cell
membranes under the same conditions which would later be
dictated by the requirements of the planar bilayer. These
conditions include an ionic gradient which is favorable for
liposome fusion, i.e. the presence of an osmotic gradient
(300 mm KC1 versus 50 mm KC1). Therefore, the low
conductance C1' channel conductance in excised membrane
patches from CFTR-expressing Sf9 cells was compared in
symmetric and asymmetric ion gradients in order to assess
the influence of these gradients on single ion conductance
(Figure 6).
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No single channel activity corresponding to that of
the low-conductance C1' channel was detected in excised
patches from CFTR-expressing Sf9 cells unless PKA (200 nM)
and Mg-ATP (1 mM) were added to the bath. With the
addition of PKA, a nonrectifying, 10.1 pS channel was
detected with 140 mM NaCl in both the patch pipette and the
bath (n=39). This observation supports the previous
description of PKA regulation of the small, nonrectifying
Cl- channel in excised patches of CHO cells expressing CFTR
(Tabcharani et al., 1991). In the presence of an ion
gradient comparable to that required for liposome fusion in
planar bilayer studies, ie. 300 mM NaCl in the bath and 50
mM NaCl in the patch pipette (n=19), the current voltage
(I-V) relationship of the PKA-regulated channel showed a
shift in reversal potential to approximately 50 mV, a
change consistent with high chloride selectivity, and an
increase in unitary conductance to 14.1 pS. This
relatively small change in conductance from 11 to 14 pS
with a two fold increase in chloride concentration suggests
that the effect of chloride concentration on unitary
conductance is nonlinear. Tabcharani and Hanrahan have
found that in excised patches from CHO cells expressing
CFTR, the low conductance Cl' channel saturates as a
function of Cl- activity with a Michaelis-Menten Km in the
range of 35 to 40 M.
In initial experiments with planar lipid bilayers, the
CFTR-containing proteoliposomes were simply added to the
cis chamber with mixing. Although indications of the
appearance of C1' channel activity were detected early on,
the fusion frequency was apparently low because the current
changes eventually found to be characteristic of this
channel in the bilayer were only observed infrequently (in
3 of 15 experiments) even in the presence of ATP and PKA.
This made it difficult to be sure of the significance of
the lack of activity under non-activating conditions.
Therefore, we sought a means of both promoting and
detecting fusion events. The nystatin fusion technique
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described just a year earlier by Woodbury and Miller (1990)
seemed as if it should suit this purpose and was attempted.
The rationale is that the ergosterol-nystatin complex which
is incorporated into phospholipid vesicles during their
formation provides non selective ionophore activity, thus
generating an ionic and osmotic gradient which promotes
fusion of the vesicles with the bilayer. When this occurs,
a transient current spike is observed providing an index of
the fusion events. Because nystatin renders essentially
all vesicles fusogenic, the channel activity observed is
representative of the whole population of vesicles. Since
we had quantified the amount of CFTR in our vesicles, this
technique provided a means of evaluating how much of it
entered the bilayer. Figure 7A shows a current tracing
containing these spikes and the coincidence of a low
conductance C1' current with one of them. On average this
occurs once in 10 and 20 spikes. Since we had calculated
that approximately one in four lipid vesicles contained a
CFTR molecule after reconstitution, it appears that 20-40%
of the purified protein molecules are capable of generating
a channel in the bilayer.
Figures 7 B, C and D show records representative of
many experiments to assess the relationships of the
properties of the channel assayed in this way to those
exhibited in the patch-clamp experiments with the cells
from which CFTR had been purified. Fusion of liposomes
without added CFTR (Figure 7B) failed to produce the
appearance of stepwise changes in current levels in the
presence of PKA and Mg-ATP (added to both cis and trans
compartment in all cases; n=6). Furthermore, fusion of
CFTR-containing liposomes without added PKA and Mg-ATP
(Figure 7C) failed to evoke the appearance of single
channel currents in 35 experiments in which fusion was
achieved. Similarly, the addition of ATP alone, prior to
PKA addition did not cause the appearance of single channel
steps. Switch-like transitions in current level were only
detected following fusion of CFTR containing liposomes in
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the presence of both PKA and ATP (Figure 7D). Single
channel events were observed in 35 of 45 experiments in
which nystatin-induced fusion spikes were observed. Hence,
in these experiments approximately 550 independent fusion
events were detected and as a consequence 35 low
conductance single channel events were detected. No single
channel currents were detected in 10 of these 45
experiments due to problems of high noise and bilayer
breakage. The mean open probability of the PKA stimulated
channel was 0.38 .13 for five experiments, a value close
to that reported by Tabcharani et al (1991) for
phosphorylation activated C1' channel in excised patches
from CFTR-expressing CHO cells of 0.41.
The current-voltage relationship of the reconstituted
CFTR protein was found to be comparable to that observed
for CFTR-expressing Sf9 membranes studied by the patch-
clamp technique in the presence of similar ionic gradients.
The slope conductance associated with the purified protein
was 11.1 pS in the presence of 300 mM KCI in the cis
compartment and 50 mM KCI in the trans compartment (Figures
8A and B). We observed no marked effect of voltage on
channel open probability, a characteristic shared with the
PKA-activated C1' channel studied in CFTR-expressing CHO
cells (Tabcharani et al, 1991). The anion versus cation
selectivity was estimated from the reversal potentials of
the I-V curves. With 300 mM KCI on the cis side and 50 mM
KCI on the trans side, one expects a reversal potential of
45 mV for an ideally anion selective channel. The
extrapolated value obtained from 6 experiments was 39 mV.
Furthermore, in the presence of the gradient (cis:trans =
300:10) it is expected that current will reverse at 86 mV
for an ideally anion selective conductance path. The
extrapolated value from four experiments was 79 mV. We
estimated using the Goldman-Hodgkin-Katz equation,
therefore, that Cl"/K+ selectivity is at least 10:1. High
chloride selectivity is another feature this channel shares
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with the CFTR-associated channel studied in CHO cells
(Tabcharani et al, 1991).
Occasionally, in 4 of 35 experiments, another
conductance level was detected in addition to the 11 pS
conductance. This relatively rare conductance level
corresponded to twice that of the predominant one and we
believe that the larger conductance reflects cooperative
gating of two 11 pS channels (Figure 8C). Cooperative
gating between different conductance states has been
described for several purified channels, including the
acetylcholine receptor (Schindler et al., 1984), bacterial
porin (Engel et al, 1985) and the ryanodine receptor (Smith
et al., 1988).
The identity of the conductance caused by purified
CFTR incorporation into lipid bilayers with that present in
cells which endogenously express CFTR, ie. T84 cells, and
that conductance conferred by CFTR expression in CHO cell
membranes, was confirmed in the experiments shown in Figure
8D. The I-V relationships of the channels activated by PKA
and ATP addition to purified CFTR, T84 and CFTR-containing
CHO membranes following fusion with the lipid bilayer are
virtually superimposable, showing similar unitary
conductances (11.1 pS, 10.0 pS and 9.23 pS respectively)
and reversal potentials (80 mV, 84 mV and 89 mV,
respectively) in the presence of a 300/10 KCI gradient.
Significantly, this PKA-activated chloride conductance was
not observed following fusion of plasma membranes prepared
from untransfected CHO cells with lipid bilayers. Only a
relatively large conductance, approximately 40 pS, chloride
channel was observed consistently when untransfected CHO
cell membranes were used, but this channel was active both
in the presence and in the absence of PKA. An identical
phosphorylation - independent channel was also observed in
CFTR-containing CHO cell membranes. This 40 pS channel is
similar with respect to its conductance and rapid kinetics
to that described by Reinhardt et al (1987) in bilayer
studies of rat colonic apical membranes.
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The inventors have demonstrated that a regulated Cl-
channel with properties similar to that observed in intact
cells can be detected in planar lipid bilayers into which
highly purified CFTR is incorporated. The CFTR protein has
5 been successfully removed from the membrane, manipulated
extensively and returned to a functional state.
To address the question of the postulated C1' channel
activity of CFTR, quantitative considerations are required.
A channel activation was observed in black lipid films
10 approximately once for every ten to twenty nystatin spikes
reflecting the fusion of 10-20 vesicles. About one in four
vesicles would contain a CFTR molecule (assuming a monomer
although we have no evidence of this). This suggested that
20-40% of the reconstituted CFTR molecules may be capable
15 of activation. This is an excellent degree of functional
reconstitution given the type of solubilization,
purification and reconstitution scheme used. However,
these data also indicate that if channel activation
required a protein other than CFTR it would have to be
present in at least one copy per 5 CFTR molecules. The one
and two dimensional gel electrophoresis data (Figures 1, 2
and 4) preclude the presence of 20% contamination.
Furthermore, the fact that the purified protein yielded
N-terminal amino sequence and overall amino acid
composition indicative of the presence of only CFTR would
argue that any contaminants which may be present must be in
very low amounts, indicating that the protein of the
invention is substantially homogeneous. Hence, we conclude
that it is very likely that regulated channel activity is
a property of the CFTR molecule itself. This invention
presents the first functional characterisation of a
purified epithelial channel.
Having the protein in isolation from others will
assist, for example, in determining specific sites of
interaction of modifying drugs such as sulfonyl ureas which
in a preliminary report have recently been claimed to
influence CFTR (Sheppard and Welsh, 1992).
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16
In Cystic Fibrosis patients, the epithelial cells of
many tissues, especially those of the lining of the
airways, either lack CFTR in the cell membrane or have non-
functional variants of the protein.
The isolation and purification of the CFTR protein, as
in the present invention, makes possible therapy for Cystic
Fibrosis patients, by restoring functional CFTR protein to
the epithelial cells of the airways of the lung.
Proteins can be incorporated into cell membranes when
they are supplied to the cell surface in association with
a suitable carrier which assists the protein to be
incorporated into the cell membrane, where it restores
function. Suitable carriers will be known to those skilled
in the art and include lipid vehicles such as the
proteoliposomes of the present invention, which fuse with
the cell surface allowing their contents to be incorporated
into the cell membrane.
The protein plus carrier is administered to the
epithelial cells to be treated by conventional means, for
example by aerosol delivery to the airways of the lung.
Additional agents may be incorporated into the
proteoliposomes to improve targeting towards a particular
tissue, for example antibodies to particular cell surface
molecules may be incorporated.
Example 1
Cell Culture
Sf9 insect cells were infected with a recombinant
Baculovirus containing the complete CFTR coding sequence as
described previously (Kartner et al., 1991). For patch-
clamp experiments cells were grown attached to plastic
tissue culture dishes in Grace's medium. For the purposes
of protein purification, cells were gown in suspension
culture. The human colonic carcinoma-derived epithelial
cell line T84 (Dharmsathaphorn et al., 1984) was grown on
a plastic substrate in 1:1 of Dulbecco's modified Eagle's
medium and F-12 culture medium, and CHO cells expressing
CFTR (Tabcharani et al, 1991) were grown in alpha modified
2061579
17
minimal essential medium. All of these culture media were
supplemented with 5 to 10% fetal bovine serum. Membrane
preparations from T84 cells were obtained as described
previously (Kartner et al, 1991) and highly purified plasma
membrane vesicles were isolated from CHO cells according to
Riordan and Ling (1979).
CFTR Purification
Sf9 cells from one liter of suspension culture were
collected 3 days after infection yielding a 5 ml pellet
which was resuspended and hypotonically swollen in 50 ml of
18 mM KC1, 5 mM sodium citrate, pH 6.8 (containing
phenylmethyl sulfonyl fluoride at 100 g/ml; aprotinin and
leupeptin at 50 g/ml). Cells were disrupted with a
Potter-Elvejham homogenizer, particulates pelleted and
treated with DNAase I(1 g per mg total protein).
Mild alkaline extraction was performed for 2 minutes
on ice with 20 volumes of 10 mM NaOH containing 0.1 mM
EDTA.
The pelleted extracted material was dissolved in 10 mM
phosphate buffer, pH 6.4 containing 2% mercaptoethanol and
2% SDS and was applied to a column (2.6 x 20 cm) of
hydroxyapatite (Biogel HT, Biorad Laboratories) which had
been preequilibrated with 10 mM phosphate buffer, pH 6.4
containing 0.15% SDS and 5 mM dithiothreitol (DTT). After
washing with 50 ml of equilibration buffer, a 100 ml linear
gradient (100 mM to 600 mM) of sodium phosphate containing
0.15% SDS and 5 mM DTT was applied at a flow rate of 0.2 ml
per minute. Elution was continued with the high phosphate
buffer for an additional 100 ml at a flow rate of 0.1 ml
per minute. Absorbance was monitored continuously at 280
nm and aliquots of each fraction were monitored for CFTR by
dot blots on nitrocellulose probed with the monoclonal
antibody M3A7. Positive fractions were further assayed by
SDS-PAGE and immunoblotting.
CFTR-containing fractions from the hydroxyapatite
column were transferred to Centricon centrifugal
microconcentrator tubes (Amicon) with a 30 kd cutoff,
2061570
18
washed with 10 mM Tris-HCI, pH 8.0 containing 100 mM NaCl
and 0.25% LiDS (lithium dodecyl sulfate) also in these
devices and again concentrated to 400 l. This volume was
applied to a Superose 6 preparative FPLC column (Pharmacia)
pre-equilibrated with this same buffer. Fractions eluted
from the Superose 6 column were monitored as with the
hydroxyapatite column.
CFTR Protein Detection and Characterization
One dimensional SDS-PAGE was according to Laemmli
(1970) using 6% acrylamide gels as described previously
(Kartner et al, 1991) or 4 to 15% gradient gels. Two
dimensional gel electrophoresis was performed according to
Dottrin et al (1979). Total proteins in either type of gel
were stained with silver (Merril et al, 1981).
Immunoblotting was as described previously employing a
monoclonal antibody (M3A7) generated against a fusion
protein containing residues 1197 - 1480 of CFTR.
CPTR Reconstitution into Phospholipid Vesicles
100 l of 15 mM HEPES, 0.5 mM EGTA, pH 7.4 containing
1 mg of a sonicated phospholipid mixture (PE:PS:PC/5:2:1)
and 2% sodium cholate was added to 100 l of 10 mM
Tris-HC1, pH 8.0 containing 100 mM NaCl, 0.25% LiDS and 5
g of purified CFTR. After a one hour incubation on ice
the mixture was dialysed at 4 C against 2 liters of the
HEPES-EDTA buffer for 5 days with daily buffer changes.
The sample was further dialysed for 3 days against daily
changes of 2 liters of the same buffer containing 150 mM
NaCl. The proteoliposomes thus obtained were sonicated for
15 seconds in a bath sonicator from Lab Supplies Co. Inc.,
Hicksville, N.Y. 11801 (Model G1128P1G) and concentrated to
100 l in a Minicon B15 concentrator (Amicon). To
introduce nystatin according to the procedure of Woodbury
and Miller (1990), this 100 l sample was mixed with 100
l of protein-free liposomes which had been prepared by
sonicating a mixture of PE:PS:PC:Ergosterol at a ratio of
5:2:1:2 (10 mg/ml) in the presence of 120 g/mi nystatin in
the HEPES-EGTA buffer containing 300 mM NaCl. The mixture
2061579
19
was frozen and thawed and sonicated for 15-20 sec. This
cycle was repeated and the final proteoliposomes either
used immediately for fusion with planar bilayers or frozen
at -85 . In the latter case aliquots were thawed and
sonicated briefly before use. The presence of intact CFTR
was
verified by exclusion from a Superose 12 column
(Pharmacia).
For estimation of their diameters, the proteoliposomes
were pipetted onto carbon formvar-coated grids, then
negatively stained with 2% aqueous uranyl acetate and
viewed and photogaphed by transmission electron microscopy.
Incorporation of CFTR into Planar Bilayers
A 10 mg/mi solution of phospholipid (PE:PS at a ratio
of 7:3) (Avanti Polar Lipids) in n-decane was painted over
a 200 m aperature in a bilayer chamber to raise
phospholipid bilayers. Bilayer formation was monitored
electrically by observing the increase in membrane
capacitance. In all experiments , bilayer capacitance was
greater than 200 pF.
The solution in the cis compartment (where
proteoliposomes were added) typically contained 300 mM KC1,
10 mM MOPS, 1 mM MgCl2 and 2 mM CaC12, pH = 6.9. The trans
solution contained 50 mM KC1, 10 mM MOPS, 1 mM MgC12 and 2
mM CaCIZ1 pH 6.9. Single channel currents were detected
with a patch-clamp amplifier, modified for bilayer studies
(Warner Instruments) and recorded following digitization
(PCM2, Medical Systems) using a VCR. For playback of
records, a 6-pole Bessel filter was used (100 Hz). Single
channel current amplitudes were determined by the
generation of amplitude histograms using pCLAMP software.
Open-state probability was determined using the same
software program and openings were defined using the 50%
threshold criterion.
Addition of 4 l of proteoliposomes preparation to the
cis compartment of the bilayer chamber, followed by
stirring (approximately 10 min) typically resulted in the
2061579
appearance of 10-20 abrupt conductance steps or spikes,
indicative of fusion of roughly 10-20 liposomes with the
lipid bilayer. The conductance steps are due to current
flow through ergosterol-dependent nystatin channels and the
5 transient nature of these steps reflects the dissociation
of the ergosterol-nystatin complex as the liposome
composition diffuses into the ergosterol-free bilayer.
Cessation of stirring prevented further liposome fusion
with no further appearance of spikes. The incorporation of
10 a channel with liposome fusion was detected as a stepwise
change in current level. Membrane potentials were
referenced to the trans compartment and C1- current from cis
to trans designated as negative.
Patch-Clamp 8tudies of CFTR-expressing cells
15 Single channel currents were recorded using conventional
procedures according to Hamill et al (Hamill et al, 1981)
with a List EPC-7 patch-clamp amplifier (Medical Systems,
Great Neck, N.Y.) Pipettes were fabricated from
borosillicate glass type 7052 ( Gamer Glass Co.) using a
20 two-stage Narishige pipette puller. The bath electrode was
a Ag-AgCl wire connected to the bathing solution via an
agar bridge. Current output was monitored on a Tektronix
oscilloscope and stored on videotape after A/D conversion
by a video adaptor (PCM 2, Medical Systems). Single
channel current records stored on video tape were
transferred to the hard disk of an EBM-AT compatible
computer using the FETCHEX program of pCLAMP (Version V)
software (Axon Inst. ) Records were sampled at 0.5-2.0 kHz.
During playback, single channel records were filtered using
a 6-pole Bessel filter set at 100 or 200 Hz low pass
frequency. Single channel current amplitudes were obtained
by examination of amplitude histograms generated using the
pCLAMP FETCHAN analysis program. The mean of the peak
amplitude was taken as a measure of the unitary current
amplitude.
solutionss In excised patch studies the standard bath
and pipette solutions contained 140 mM NaCl, 1 mM MgC12, 2
2061579
21
mM CaC121 10 mM glucose and 10 mM MES, pH 6.3. In some
studies, the pipette solution contained 50 mM NaC1 plus
sucrose (added to adjust osmolarity to 280 mOsm) and the
bath solution contained 300 mM NaCl. Experiments were
performed at 22-25 C.
2061579
22
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27
Table I
Protein Recovery during CPTR Purification
Step Total protein Enrichment factor*
(mg)
Particulate pellet 300 1
After alkali extraction 105 2.9
After hydroxyapatite 1.0 300
After Superose 6 0.5 600
Starting material was a one liter culture containing
approximately 5 x 109 cells.
* assuming no loss of CFTR.
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28
Table II
Amino Acid Composition
Residues Predicted* Determined Ratio
Determined/
Predicted
Asx 113 100 0.88
Glx 160 154 0.96
Ser 122 127 1.04
Gly 84 84 1.00
His 24 24.4 1.01
Arg 78 76.4 .98
Thr 83 86.6 1.04
Ala 83 83 1.00
Pro 45 42 0.93
Tyr 40 nd
Val 89 89.3 1.00
Met 38 36 0.95
Cys 18 20.8 1.15
Ile 120 113 0.94
Leu 18 181.2 0.98
Phe 84 82.8 0.99
Lys 92 96.8 1.05
* from sequence
nd - not determined
1.5 g of protein was hydrolysed with 6N HC1, PITC
derivatized and separated by HPLC on a PICOTAG column
(Waters Associates).
