©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Recombinant Cystic Fibrosis Transmembrane Conductance Regulator from Chinese Hamster Ovary and Insect Cells (*)

Catherine R. O'Riordan (§) , Amy Erickson , Christine Bear (1)(¶), Canhui Li (1), Partha Manavalan , Kathryn X. Wang , John Marshall , Ronald K. Scheule , John M. McPherson , Seng H. Cheng , Alan E. Smith

From the (1)Genzyme Corporation, Framingham, Massachusetts 01701-9322 Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, -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.


INTRODUCTION

Cystic fibrosis (CF),()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).

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) 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.

The most common CF-associated mutation, a deletion of the residue phenylalanine at position 508 (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.

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.()


EXPERIMENTAL PROCEDURES

Materials

Phosphatidylethanolamine, phosphatidylcholine (PC), phosphatidylserine, -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 KHPO was applied at a flow rate of 2 ml/min. CFTR eluted from the column at approximately 60-85 mM KHPO.

Purification of CFTR from Baculovirus-infected Sf9 Cells

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 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

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.).

ELISA

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.

Photoaffinity Labeling of CFTR

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-[-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

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 KHPO, 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

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).

Planar Lipid Bilayer Studies

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, 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.


RESULTS

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.

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).


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.

Several chromatographic methods were examined for their ability to purify CFTR from the -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.

Greater than 95% of the -lysoPC-solubilized CFTR bound to DEAE-Sepharose when chromatographed in 10 mM KHPO, pH 7.5. Fig. 2shows that when a linear gradient of 10-150 mM KHPO was applied to the resin, CFTR eluted over a broad range beginning at approximately 60 mM KHPO. 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 KHPO 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.

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).


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.




DISCUSSION

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-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.

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 -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.

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 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.

The immunoaffinity-purified CFTR from CHO cells also exhibited another Cl 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).

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 (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.

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.

  
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



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Genzyme Corp., One Mountain Rd., Framingham, MA 01701-9322. Tel.: 508-872-8400 (ext. 2448); Fax: 508-872-9080.

A Medical Research Council (MRC) Scientist, supported by operating grants from the MRC (Canada).

The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibro-sis transmembrane conductance regulator; CHO, Chinese hamster ovary; PC, phosphatidylcholine; ELISA, enzyme-linked immunosorbent assay; -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).

R. K. Scheule, R. Bagley, A. Erickson, K. X. Wang, S. L. Fang, C. Vaccaro, C. O'Riordan, S. H. Cheng, and A. E. Smith, submitted for publication.

T. Bond, unpublished results.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Altman, F., Kornfeld, G., Dalik, T., Staudacher, E., and Glöss, T. (1993) J. Glycobiology3, 619-625
  2. Anderson M. P., and Welsh, M. J. (1992) Science257, 1701-1704 [Medline] [Order article via Infotrieve]
  3. Anderson, M. P., Berger, H. A., Rich, D. P., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1991) Cell67, 775-784 [Medline] [Order article via Infotrieve]
  4. Arion, W. J., and Racker, E. (1970) J. Biol. Chem.245, 5186-5194 [Abstract/Free Full Text]
  5. Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M., and Riordan, J. R. (1992) Cell68, 809-818 [Medline] [Order article via Infotrieve]
  6. Bell, C. L., and Quinton, P. M. (1993) Am. J. Physiol.264, C925-C931
  7. Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Mulligan, R., Smith, A. E., and Welsh, M. J. (1991) J. Clin. Invest.88, 1422-1431 [Medline] [Order article via Infotrieve]
  8. Boat, T., Welsh, M. J., and Beaudet, A. (1989) in The Metabolic Basis of Inherited Diseases (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds) pp. 2649-2860, McGraw-Hill, New York
  9. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., O'Riordan, C. R., and Smith, A. E. (1990) Cell63, 827-834 [Medline] [Order article via Infotrieve]
  10. Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991) Cell66, 1027-1036 [Medline] [Order article via Infotrieve]
  11. Dalemans, W., Barby, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G., Pavirani, A., Lecocq, J., and Lazdunski, M. (1991) Nature354, 524-528
  12. Denning, G. M., Ostedgaard, L. S., and Welsh, M. (1992a) J. Cell Biol.118, 551-559 [Abstract]
  13. Denning, G. M., Anderson, M. P., Amara, J. F. Marshall, J., Smith, A. E., and Welsh M. J. (1992b) Nature358, 761-764 [CrossRef][Medline] [Order article via Infotrieve]
  14. Drumm, M. L., Wilkinson, D. J., Smit, L. S., Worrell, R. T., Strong, T. V., Frizzell, R. A., Dawson, D. C., and Collins, F. S. (1991) Science254, 1797-1799 [Medline] [Order article via Infotrieve]
  15. Engvall, E., and Perlman, P. (1971) Immunochemistry8, 871-875 [CrossRef][Medline] [Order article via Infotrieve]
  16. Graham, F. L., and van der Eb, A. J. (1973) Virology52, 456-467 [Medline] [Order article via Infotrieve]
  17. Gregory, R. J., Cheng, S. H., Rich, D. P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K. W., Welsh, M. J., and Smith, A. E. (1990) Nature347, 382-386 [CrossRef][Medline] [Order article via Infotrieve]
  18. Gunderson, K. L., and Kopito, R. R. (1994) J. Biol. Chem.269, 19349-19353 [Abstract/Free Full Text]
  19. Hsieh, P., and Robbins, P. W. (1984) J. Biol. Chem.259, 2375-2382 [Abstract/Free Full Text]
  20. Illsley, N. P., and Verkman, A. S. (1987) Biochemistry26, 1215-1219 [Medline] [Order article via Infotrieve]
  21. Jarvis, D. L., and Summers, M. D. (1989) Mol. Cell. Biol.9, 214-223 [Medline] [Order article via Infotrieve]
  22. Jones, O. T., Earnest, J. P., and MNamee, M. G. (1986) in Solubilization and Reconstitution of Membrane Proteins in Biological Membranes: A Practical Approach (Findlay, J., ed) IRL Press
  23. Kagawa, Y., and Racker, E. (1971) J. Biol. Chem.246, 5477-5487 [Abstract/Free Full Text]
  24. Kartner, N., Hanrahan, J. W., Jensen, T. J., Naismith, A. L., Sun, S., Ackerly, C. A., Reyers, E. F., Tsui, L.-C., Rommens, J. M., Bear, C., and Riordan, J. R. (1991) Cell64, 681-691 [Medline] [Order article via Infotrieve]
  25. Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A.. L., and Riordan, J. R. (1992) Nature Genet.1, 321-327 [Medline] [Order article via Infotrieve]
  26. Luckow, V. A., and Summers, M. D. (1988) BioTechniques6, 47-55
  27. Marshall, J., Fang, S., Ostedgaard, L. S., O'Riordan, C. R., Ferrara, D., Amara, J. F., Hoppe, H., IV, Scheule, R. K., Welsh, M. J., Smith, A. E., and Cheng, S. H. (1994) J. Biol. Chem.269, 2987-2995 [Abstract/Free Full Text]
  28. Miller, L. K. (1988) Annu. Rev. Microbiol.42, 177-199 [CrossRef][Medline] [Order article via Infotrieve]
  29. Quinton, P. M., and Reddy, M. M. (1992) Nature360, 79-81 [CrossRef][Medline] [Order article via Infotrieve]
  30. Rich, D. P., Anderson, M. P., Gregory, R. G., Cheng, S. H., Paul, S., Jefferson, D. M., McCann, J. D., Klinger, K. W., Smith, A. E., and Welsh, M. J. (1990) Nature347, 358-363 [CrossRef][Medline] [Order article via Infotrieve]
  31. Riordan, J., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzi, M. C., Collins, F. S., and Tsui, L.-C. (1989) Science245, 1066-1073 [Medline] [Order article via Infotrieve]
  32. Tabcharani, J. A., Chang, X.-B., Riordan, J. R., and Hanrahan, J. W. (1991) Nature352, 628-631 [CrossRef][Medline] [Order article via Infotrieve]
  33. Tilly, B. C., Winter, M., Ostedgaard, L. S., O'Riordan, C., Smith, A. E., and Welsh, M. J. (1992) J. Biol. Chem.267, 9470-9473 [Abstract/Free Full Text]
  34. Travis, S. M., Carson, M. R., Rirs, D. R., and Welsh, M. J. (1993) J. Biol. Chem.268, 15336-15339 [Abstract/Free Full Text]
  35. Tsui, L. C. (1992) Hum. Mutat.1, 197 [Medline] [Order article via Infotrieve] -203
  36. Urlaub, G., and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. U. S. A.77, 4216-4220 [Abstract]
  37. Welsh, M. J., and Smith, A. E. (1993) Cell73, 1251-1254 [Medline] [Order article via Infotrieve]
  38. Welsh, M. J., Anderson, M. P., Rich, D. P., Berger, H. A., Denning, G. M., Ostedgaard, L. S., Sheppard, D. N., Cheng, S. H., Gregory, R. J., and Smith, A. E. (1992) Neuron5, 821-829
  39. Wigler, M., Silverstein, S., Lee, L. S., Pellcier, A., Cheng, V., and Axel, R. (1977) Cell11, 223-232 [Medline] [Order article via Infotrieve]
  40. Woodbury, D. J., and Miller, C. (1990) Biophys. J.58, 833-839 [Abstract]

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