# US 5,215,963 A · Solubilization and purification of the active gastrin releasing peptide receptor

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- Number: US 5,215,963 A
- Document ID: US5215963A
- Status: Granted
- Kind: A
- Application number: 07/426,150
- Filed: 1989-10-24
- Granted: 1993-06-01
- Earliest claimed: 1989-10-24
- Assignees: Berlex Laboratories, Inc.
- Inventors: Richard I. Feldman; James M. Wu; Elaina Mann; Anne Larocca; James C. Jenson
- Web page: https://patentgrep.com/patent/US5215963A

## CPC

- G01N 33/5752: of the lungs
- C07K 14/70571: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- C07K 16/286: {against neuromediator receptors, e.g. serotonin receptor, dopamine receptor}
- G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds}
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- G01N 2333/5758: Gastrin releasing peptide
- G01N 2333/70571: for neuromediators, e.g. serotonin receptor, dopamine receptor
- G01N 2333/72: for hormones
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

A procedure for the preparation of the solubilized and purified gastrin releasing peptide receptor, in an active form, from a gastrin releasing peptide receptor source such as Swiss 3T3 fibroblasts.

## Claims

### Claim 1

A method for solubilizing the gastrin releasing peptide receptor in an active form comprising the steps of: (1) preparing membranes from a gastrin releasing peptide receptor source;
(2) washing said membranes and suspending said membranes in a buffer;
(3) solubilizing the gastrin releasing peptide receptor by incubating a culture of said membranes in a culture medium to which a detergent and a soluble cholesteryl ester has been added; and
(4) adjusting the detergent concentration of said solubilized gastrin releasing peptide receptor.

### Claim 2 (depends on 1)

The method of claim 1 where said detergent and soluble cholesteryl ester are added to the incubating mixture in step (3) at the same time.

### Claim 3 (depends on 1)

The method of claim 1 wherein said detergent is added to the incubating mixture in step (3) prior to the addition of the soluble cholesteryl ester.

### Claim 4 (depends on 1)

The method of claim 1 wherein said gastrin releasing peptide receptor source is a eukaryotic or prokaryotic host expressing recombinant gastrin releasing peptide receptor.

### Claim 5 (depends on 1)

The method of claim 1 wherein said gastrin releasing peptide source is a mammalian cell line.

### Claim 6 (depends on 5)

The method of claim 5 wherein said cell line is human.

### Claim 7 (depends on 5)

The method of claim 5 wherein said cell line is mouse Swiss 3T3 fibroblasts.

### Claim 8 (depends on 1)

The method of claim 1 wherein said detergent is 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate.

### Claim 9 (depends on 1)

The method of claim 1 wherein said soluble cholesteryl ester is cholesteryl hemisuccinate.

### Claim 10 (depends on 1)

The method of claim 1 wherein the weight ratio of detergent to soluble cholesteryl ester is within the range of about 200:1 to 5:2.

### Claim 11 (depends on 10)

The method of claim 10 wherein said ratio is about 10:1.

### Claim 12 (depends on 1)

The method of claim 1 wherein the concentration of detergent in step (3) is within the range of about 0.4% to 3.0% (w/v).

### Claim 13 (depends on 1)

The method of claim 1 wherein the concentration of detergent in step (4) is within the range of about 0.025% to 0.2% (w/v).

### Claim 14

A method for solubilizing and purifying the gastrin releasing peptide receptor in an active form comprising the steps of:
(1) preparing membranes from a gastrin releasing peptide receptor source;
(2) washing said membranes and suspending said membranes in a buffer;
(3) solubilizing the gastrin releasing peptide receptor by incubating a culture of said membranes in a culture medium to which a detergent and a soluble cholesteryl ester has been added, forming a soluble extract;
(4) precipitating the gastrin releasing peptide receptor from the soluble extract of step (3), collecting the precipitate by centrifugation and resuspending said precipitate in a buffer containing said detergent and soluble cholesteryl ester, forming a soluble extract;
(5) applying the soluble extract of step (4) to a wheat germ agglutinin affinity column forming an eluate;
(6) further fractionating the wheat germ agglutinin column eluate from step (5) on a gastrin releasing peptide affinity column, forming an eluate; and
(7) dialyzing the gastrin releasing peptide affinity column eluate from step (6) and applying said eluate to a second gastrin releasing peptide affinity column.

### Claim 15 (depends on 14)

The method of claim 14 which further comprises a gel filtration step following step (7).

### Claim 16 (depends on 14)

The method of claim 14 where said detergent and soluble cholesteryl ester are added to the incubating mixture in step (3) at the same time.

### Claim 17 (depends on 14)

The method of claim 14 wherein said detergent is added to the incubating mixture in step (3) prior to the addition of the soluble cholesteryl ester.

### Claim 18 (depends on 14)

The method of claim 14 wherein said gastrin releasing peptide receptor source is a eukaryotic or prokaryotic host expressing recombinant gastrin releasing peptide receptor.

### Claim 19 (depends on 14)

The method of claim 14 wherein said gastrin releasing peptide source is a mammalian cell line.

### Claim 20 (depends on 19)

The method of claim 19 wherein said cell line is human.

### Claim 21 (depends on 19)

The method of claim 19 wherein said cell line is mouse Swiss 3T3 fibroblasts.

### Claim 22 (depends on 14)

The method of claim 14 wherein said detergent is 3-[(3-cholamidopropyl)dimethylamaonio]-1-propane sulfonate.

### Claim 23 (depends on 14)

The method of claim 14 wherein said soluble cholesteryl ester is cholesteryl hemisuccinate.

### Claim 24 (depends on 14)

The method of claim 14 wherein the weight ratio of detergent to soluble cholesteryl ester is within the range of about 200:1 to 5:2.

### Claim 25 (depends on 24)

The method of claim 24 wherein said ratio is about 10:1.

### Claim 26 (depends on 14)

The method of claim 14 wherein the concentration of detergent in step (3) is within the range of about 0.4% to 3.0% (w/v).

### Claim 27 (depends on 14)

The method of claim 14 wherein the concentration of detergent in step (4) is within the range of about 0.025% to 0.2% (w/v).