WO1995007453A1

WO1995007453A1 - A method for purifying a membrane-associated protein

Info

Publication number: WO1995007453A1
Application number: PCT/US1994/009948
Authority: WO
Inventors: Catherine R. O'riordan; Amy Erickson
Original assignee: Genzyme Corp
Current assignee: Genzyme Corp
Priority date: 1993-09-08
Filing date: 1994-09-02
Publication date: 1995-03-16
Anticipated expiration: 1996-03-08
Also published as: AU7644494A

Abstract

The present invention provides a method for purifying a membrane associated protein, e.g., cystic fibrosis transmembrane conductance regulator (hereinafter CFTR) in a functional form. The method involves contacting a membrane-associated protein-membrane fraction complex with a detergent forming a solubilized complex and chromatographically isolating the membrane-associated protein from the solubilized complex in a functional form. The functional form of the purified membrane-associated protein of the present invention preferably is sufficiently pure to allow its introduction into humans for therapeutic purposes.

Description

A METHOD FOR PURIFYING A MEMBRANE- ASSOCIATED PROTEIN

Related Applications This application is a continuation-in-part application of USSN

08/114,950 filed on August 27, 1993, entitied ANTIBODIES SPECIFIC FOR CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR AND USES THEREFOR by Cheng et al. which is a continuation-in-part of USSN 08/087,132, filed July 2, 1993, which is a continuation application of USSN 07/613,592, filed November 15, 1990, which is in turn a continuation-in-part application of USSN 07/589,295, filed September 27, 1990, which is a continuation-in-part application of USSN 07/488,307, filed March 5, 1990. This application is also related to the subject matter described in co-pending application USSN 07/985,478 filed December 2, 1992. The contents of all of the above co-pending patent applications are incorporated herein by reference. Definitions of language or terms not provided in the present application are the same as those set forth in the copending applications. Any reagents or materials used in the examples of the present application whose source is not expressly identified also is the same as those described in the copending application, e.g., ΔF508 CFTR gene and CFTR antibodies.

Background of the Invention

Cystic Fibrosis (CF) is the most common fatal genetic disease in humans (Boat, T. et al. Cystic fibrosis. In: The Metabolic Basis of Inherited Disease, C. Scriver, A. Beaudet, W. Sly, and D. Valle, eds.

(McGraw Hill, New York, 1989), 2649-2860). Based on both genetic and molecular analysis, a gene associated with CF was isolated as part of 21 individual cDNA clones and its protein product predicted (Kerem, B-S. et al. Science 245:1073-1080 (1989); Riordan, J. et al. Science 245:1066-1073 (1989); Rommens, J.H. et al. Science 245:1059-1065 (1989)).

USSN 07/488,307 describes the construction of the gene into a continuous strand, expression of the gene as a functional protein and confirmation that mutations of the gene are responsible for CF. (See also Gregory, R.J. et al. Nature 347:382-386 (1990); Rich, D.P. et al. Nature 347:358-363 (1990)). The copending patent application also discloses experiments which showed that proteins expressed from wild type but not a mutant version of the cDNA complemented the defect in the cAMP regulated chloride channel shown previously to be characteristic of CF.

The protein product of the CF associated gene is called the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan, J. et al. Science 245:1066-1073 (1989)). CFTR is a protein of approximately 1480 amino acids made up of two repeated elements, each having six transmembrane segments and a nucleotide binding domain. The two repeats are separated by a large, polar, so-called R-domain containing multiple potential phosphorylation sites. Based on its predicted domain structure, CFTR is a member of a class of related proteins which includes the multi- drug resistant (MDR) P-glycoprotein, bovine adenyl cyclase, the yeast STE6 protein as well as several bacterial amino acid transport proteins (Riordan, J. et al. Science 245:1066-1073 (1989); Hyde, S.C. et al. Nature 346:362-365 (1990). Proteins in this group, characteristically, are involved in pumping molecules into or out of cells. CFTR has been postulated to regulate the outward flow of anions from epithelial cells in response to phosphorylation by cyclic AMP- dependent protein kinase or protein kinase C (Riordan, J. et al. Science 245:1066-1073 (1989); Frizzell, R.A. et al. Science 233:558-560 (1986); Welsh, M.J. and Liedtke, CM. Nature 322:467 (1986); Li, M. et al. Nature 331:358-360 (1988); Hwang, T-C. et al. Science 244;1351-1353 (1989); Li, M. et al. Science 244:1353-1356 (1989)). Difficulties can be encountered when attempting to purify CFTR or other membrane-associated proteins because a purification process may not result in a functional protein.

Summary of the Invention The present invention provides a method for purifying a membrane associated protein, e.g., cystic fibrosis transmembrane conductance regulator (hereinafter CFTR), in a functional form. The method involves contacting a membrane-associated protein-membrane fraction complex with a detergent forming a solubilized complex and chromatographically isolating the membrane-associated protein from the solubilized complex in a functional form. The functional form of the purified membrane-associated protein of the present invention preferably is sufficiently pure to allow its introduction into humans for therapeutic purposes.

Detailed Description The present invention pertains to a method involving contacting a membrane-associated protein-membrane fraction complex with a detergent forming a solubilized complex and chromatographically isolating the membrane-associate protein from the solubilized complex in a functional form. The language "membrane-associated protein" is intended to include transmembrane proteins, peripheral membrane proteins, and integral membrane proteins. Transmembrane proteins extend across the membrane. Peripheral membrane proteins are bound to one or the other face of the membrane generally through interactions with, for example, other membrane- associated proteins. Peripheral membrane proteins can also be associated with the membrane by attachment to a fatty acid chain inserted into the membrane or by attachment to an oligosaccharide which is in turn attached to a fatty acid chain inserted into the membrane. Integral membrane proteins are generally transmembrane proteins linked to a fatty acid chain in the membrane. Examples of transmembrane proteins include ion channels, particularly chloride ion channels such as cystic fibrosis transmembrane conductance regulator (CFTR). The language "detergent" is intended to include surface active agents capable of forming solubilized complexes of the membrane-associated protein-membrane fraction complex. Several different criteria are used for choosing a detergent suitable for solubilizing the complex. For example, one property considered is the ability of the detergent to solubilize the CFTR within the membrane fraction at minimal denaturation of the membrane- associated protein allowing for the activity or functionality of the membrane- associated protein to return upon reconstitution of the protein. Another property considered when selecting the detergent is the critical micells concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution. A third property considered when selecting a detergent is the hydrophobicity of the detergent. Typically, membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g. the triton series, would be useful for solubilizing the hydrophobic proteins. Another property important to a detergent can be the capability of the detergent to remove the CFTR with minimal protein-protein interaction facilitating further purification. A fifth property of the detergent which is considered is the charge of the detergent. For example, if it is desired to use ion exchange resins in the purification process then the preferably detergent would be an uncharged detergent. Examples of detergents which are useful within the present invention include CHAPS and CHAPS 0 (preferably in the presence of 0.5M NaCl), Digitonin, Octylglucoside, HECAMEG, Triton X- 100 and Triton X-114, sodium cholate, dodecylmaltoside, sucrose monolaurate, Deoxy BIG-CHAP α-lyso Phosphatidylcholine (α-lyso PC), α- lyso Palmitoyl Phosphatidylcholine (α-lyso PPC), and α-lyso myristoyl Phosphatidylcholine. The preferred detergent of the present invention for CFTR is α-lyso PC which solubilizes 80 percent CFTR from the membrane fraction and approximately 50 percent total cell protein. It should be understood that the solubilization effect of the detergent can be increased or adjusted by adjusting other parameters, e.g.increasing the salt concentration.

The language "forming a solubilized complex" is intended to include complexes wherein the membrane-associated protein is not entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be chromatographically isolated from the membrane fraction. The membrane-associated protein can be completely solubilized or separated from the membrane fraction complex but does not have to be completely separated and can be separated to the extent sufficient to allow chromatographic isolation.

The language "membrane fraction" is intended to include the portion of the membrane in association with the membrane associated protein after lysis. The membrane fraction includes the membrane vesicle.

The language "chromatographically isolating" is intended to include techniques capable of isolating the membrane-associated protein based upon principles of chromatography. Examples of types of such art- recognized chromatographic techniques include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity. Examples of materials used within hydrophobic interaction chromatographic techniques include phenyl sepharose, butyl sepharose and artificial membrane technology. An example of a material used in a lectin affinity chromatographic technique is wheat germ agglutin sepharose. The protein being separated in the lectin affinity chromatographic technique can be eluted from the material using N-acetyl glucosamine. Examples of materials used in ion-exchange chromatographic techniques include S -sepharose, Q- sepharose, DEAE-sepharose and CM-sepharose. Examples of materials used in dye affinity techniques include resins selected from the group consisting of basilene blue dye, piski dye and green dye. Examples of materials used in an immunoaffinity chromatographic technique include monoclonal antibodies directed towards both the R-domain and the C-terminus of the membrane-associated protein. For example, a monoclonal antibody directed towards the R-domain of CFTR is monoclonal antibody 13-1 and a monoclonal antibody directed toward the C-terminus of CFTR is monoclonal antibody 24-1. These monoclonal antibodies were previously described in a co-pending application cited supra. The purification process of the present invention can also contain optional steps such as a gel filtration step subsequent to the chromatographic isolation step and a stripping step prior to the contacting step. Gel filtration is art recognized and an example of a material which can be used in the gel filtration step is superdex 200 HR 10/30 (Pharmacia) as described in the examples below. The alkali stripping step is also described in detail in the examples set forth below.

The present invention also pertains to purified membrane associated proteins produced by the methods of the present invention. The purified membrane-associated proteins preferably are sufficiently pure to allow their introduction into mammals, especially humans. This language is intended to include a level selected by the appropriate regulatory agency for the use of the membrane-associated protein and the various steps of the methods of the present invention can be varied to achieve the desired or required purity. For example, the FDA can require at least a 90 percent purity on some human injectables. The purified membrane-associated protein product of the present invention preferably has a purity of at least about 50 percent, more preferably at least about 70 percent and most preferably at least about 80 percent.

It should be understood that various combinations of the chromatographic material and detergent may be desired for a particular membrane-associated protein or a particular detergent may be preferably when using a particular chromatographic material. Examples of some useful combinations are set forth in Table 1 below.

Table 1

Membrane-Associated Chromatographic Protein Material Detergent

A) CFTR S-Sepharose CHAPSO (Pharmacia) Lyso-PC

B) CFTR Q-Sepharose Lyso PC (Pharmacia) Mega sucrose monolaurate deoxy BIG- CHAP

C) CFTR DEAE-Sepharose Lyso-PC (Pharmacia)

D) CFTR CM-Sepharose Sucrose (Pharmacia) monolaurate

E) CFTR Phenyl-Sepharose Triton X-100 (Pharmacia)

F) CFTR Phenyl-Sepharose SDS (ISS)

G) CFTR Butyl-Sepharose CHAPSO (Pharmacia)

H) CFTR Mimetic Column- CHAPSO IAM Chromatography Triton X-100 (Regis Chemicals)

I) CFTR Basilene Blue Resin CHAPSO

J) CFTR Piksi Dye Affinity Resin Triton X-100 (ISS)

K) CFTR Green Dye Resin Triton X-100

L) CFTR Wheat Germ Lectin CHAPSO (Pharmacia) The present invention is further illustrated by the following examples which in no way should be construed as being further limiting. The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLES

Example 1 Purification of CFTR from CHO-CFTR Cells Using α-lvso Phosphatidylcholine and A Monoclonal Antibody 13-1 Immunoaffinity Resin

Preparation of the Membrane Fractions

CHO cells stably expressing CFTR (hereinafter CHO CFTR cells) were prepared and pelleted as described previously (Tilly et al. (1992) The Journal of Biological Chemistry, Vol. 267, No. 14, pp. 9470-73; Anderson et al. (1991) Science, 251, pp. 679-682). The CHO CFTR cells were grown in spinners (8L) on DE52 microcarriers (sold by Pharmacia). The cells were pelleted from the cell culture by centrifuging (lOOOxg) for five minutes. The cell pellets were washed twice with phosphate buffered saline (PBS) and the wash solution was discarded. The pellets were resuspended in a hypotonic lysis buffer (10 mM NaCl, 20 mM Tris HC1, 1 mM EDTA, 2 mM MgCl₂) on ice for thirty minutes. Protease inhibitors included with the hypotonic lysis buffer were as follows: DTT (5 mM), benzamidine (10 mM), PMSF (0.5 mM), leupeptin (1 uM), Pepstatin A (1 uM) and Aprotinin (20 ug/ml). The suspension was passed through a microfluidizer at 2000-3000 psi and centrifuged (1000 xg) for ten minutes. The supernatant was separated and centrifuged (10,000 xg) for an additional twenty-five minutes. Again, the supernatant was separated and centrifuged (100,000 xg) for one hour. The final pellet was the crude membrane fraction containing the CFTR (hereinafter the CFTR-membrane fraction complex). The CFTR-membrane fraction complex was resuspended in a solution (150 mM NaCl, 50 mM Tris HCl, 1 mM EDTA, 10% glycerol (pH 7.5)) and then subjected to the stripping process described below.

Alkali Stripping of CFTR-Membrane Fraction Complex

The alkali stripping is an optional step which removes the peripherally bound membrane proteins from the complex. The resulting complex is enriched for CFTR as this procedure removes a large percentage of total membrane protein, e.g. 60 percent, but does not remove the CFTR or membrane-associated protein.

The CFTR-membrane fraction complex was diluted with ten volumes of 10 mM EDTA (pH 10.0) and allowed to sit on ice for two minutes. The stripped complex was collected via centrifugation (100,000 xg) of the diluted solution for approximately sixty minutes forming a stripped CFTR-membrane fraction complex.

Solubilization of the Stripped CFTR-Membrane Fraction Complex and Purification of CFTR from the Solubilized Complex Using MAb 13-1 Immunoaffinitv Resin: Peptide Elution

The stripped complex was diluted (4 mg/ml) in a solubilization buffer (150 mM KCl; 50 mM Tris HCl; 1 mM EDTA; 10% glycerol; 1.5% α-lyso PC (pH 7.5)) and rotated for one hour at 4°C. Subsequently, the solution was centrifuged at 4°C for one hour at 1000,000 xg (40,000 rpm in A641 rotor). The supernatant was saved and rocked with approximately 15 mis. of MAb 13-1 hydrazide resin overnight at 4°C in a batch process. The resin was washed with 100 mis of a wash buffer (150 mM NaCl; 50 mM Tris HCl; 1 mM EDTA, 1% Cholate (pH 8.0)) also in a batch process. The resin was gently resuspended in a column with 10 mis of elution buffer (150 mM KCl; 50 mM Tris HCl; 1 mM EDTA; 10% glycerol; 5 mM peptide for MAb 13-1: SDEPLERRLS-NH₂ MW=1189.9; 0.5% cholate. Immediately before use 1 mM Pefabloc, Aprotinin 20 ug/ml, 10 mM benzamidine, 5 ug/ml Pepstatin A, 5 ug/ml Leupeptin was added) and rocked for thirty minutes to equilibrate. The resin column was completely drained and the fraction was collected. This process was repeated four times using ten to fifteen minute incubations each time collecting the fractions. The column was washed with approximately 50 mis of storage buffer (150 mM NaCl, 25 mM Tris HCl, 1 mM EDTA and 0.02% NaN₃) and stored at 4°C. The collected fractions containing immunoaffinity purified CFTR (approximately 50 percent pure) were pooled and concentrated to 500 ul using a Centricell concentrating device. An aliquot (200 ul) of this concentrate was applied to a Superdex resin and the purified CFTR collected from the Superdex resin was approximately 80 percent pure. Approximately 300 mgs of the CFTR- membrane fraction complex yielded approximately 1 mg of 80 percent pure CFTR.

Example 2 Purification of CFTR from CHO-CFTR Cells Using α-lvso Phosphatidylcholine and A Monoclonal Antibody 24-1 Immunoaffinity Resin

This example was conducted as described in Example 1 above with the exception that a MAb 24-1 hydrazide resin was used in place of the MAb 13-1 hydrazide resin and 5 mM peptide for MAb 24-1: VQDTRL MW: 726.8 was used in the elution buffer in place of the peptide for MAb 13-1. The final CFTR production was 50 percent pure and the yield was similar to that described in Example 1.

Example 3 Purification of CFTR from CHO-CFTR Cells Using α-lvso Phosphatidylcholine and A Monoclonal Antibody 13-1 Immunoaffinity Resin and High pH Elution

This example is conducted as described in Example 1 above with the exception that a high pH elution buffer (150 mM NaOH, 10% glycerol, 0.5% Cholate (pH 11.0)) is used in the immunoaffinity purification step. The column is eluted at 1 ml/minute and the fractions (5 ml) are collected. The fractions are immediately neutralized by adding 1 M Tris HCl (250 ul) to the collection tubes prior to collecting the fractions (final concentration = 50 mM). The neutralized fractions are further purified as described in Example 1.

Example 4 Purification of CFTR from CHO-CFTR Cells Using α-lvso Phosphatidylcholine and a Monoclonal Antibody 24-1 Immunoaffinity Resin and High pH Elution

This example is conducted as described in Example 2 above with the exception that a high pH elution buffer (150 mM NaOH, 10% glycerol, 0.5% Cholate (pH 11.0)) is used in the immunoaffinity purification step. The column is eluted at 1 ml/minute and the fractions (5 ml) are collected. The fractions are immediately neutralized by adding 1 M Tris HCl (250 ul) to the collection tubes prior to collecting the fractions (final concentration = 50 mM). The neutralized fractions are further purified as described in Example 2.

EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for purifying a membrane-associated protein, comprising contacting a membrane-associated protein-membrane fraction complex with a detergent forming a solubilized complex; and chromatographically isolating the membrane-associated protein from the solubilized complex in a functional form.

2. The method of claim 1 wherein the membrane-associated protein is an ion channel.

3. The method of claim 2 wherein the ion channel is a chloride channel.

4. The method of claim 1 wherein the membrane-associated protein is CFTR.

5. The method of claim 1 further comprising stripping the membrane- associated protein-membrane fraction complex with alkali prior to the contacting step forming a stripped complex.

6. The method of claim 1 wherein the membrane-associated protein is chromatographically isolated using a hydrophobic interaction chromatographic technique.

7. The method of claim 6 wherein the hydrophobic interaction chromatographic involves a material selected from the group consisting of phenyl sepharose, butyl sepharose, and artificial membrane technology.

8. The method of claim 1 wherein the membrane-associated protein is chromatographically isolated using a lectin affinity chromatographic technique.

9. The method of claim 1 wherein the lectin affinity chromatographic technique involves wheat germ agglutin sepharose.

10. The method of claim 9 further comprising eluting the membrane- associated protein from the wheat germ agglutin sepharose using N-acetyl glucosamine.

11. The method of claim 1 wherein the membrane-associated protein is chromatographically isolated using an ion-exchange technique.

12. The method of claim 11 wherein the ion-exchange technique involves a material selected from the group consisting of S-sepharose, Q-sepharose,

DEAE- sepharose, and CM-sepharose.

13. The method of claim 1 wherein the membrane- associated protein is chromatographically isolated using a dye affinity technique.

14. The method of claim 1 wherein the dye affinity technique involves a resin selected from the group consisting of basilene blue dye, piksi dye, and green dye.

15. The method of claim 1 wherein the membrane-associated protein is chromatographically isolated using an immunoaffinity chromatographic technique.

16. The method of claim 15 wherein the immunoaffinity chromatographic technique involves monoclonal antibodies directed towards both the R- domain and C-terminus of the membrane-associate protein.

17. The method of claim 15 further comprising a gel filtration step subsequent to the chromatographic isolation step.

18. The method of claim 4 wherein the membrane-associated protein is chromatographically isolated using an immunoaffinity chromatographic technique.

19. The method of claim 18 wherein the immunoaffinity chromatographic technique involves monoclonal antibodies directed towards both the R- domain and C-terminus of the membrane-associate protein.

20. The method of claim 19 wherein the monoclonal antibody directed towards the R-domain is mAb 13-1 and the monoclonal antibody directed towards the C-terminus is mAb 24-1.

21. A purified membrane-associated protein produced by the method of claim 1.

22. The purified membrane-associated protein of claim 21 being sufficiently pure to allow its introduction into humans.

23. The purified membrane- associated protein produced of claim 21 having a purity of at least about 50 percent.

24. The purified membrane- associated protein of claim 23 having a purity of at least about 70 percent.

25. The purified membrane-associated protein of claim 23 having a purity of at least about 80 percent.