# US 2014/0194595 A1 · Method and Composition for Crystallizing G Protein-Coupled Receptors

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- Number: US 2014/0194595 A1
- Document ID: US20140194595A1
- Status: Published application
- Kind: A1
- Application number: 14/133,279
- Filed: 2013-12-18
- Published: 2014-07-10
- Earliest claimed: 2007-10-17
- Assignees: The Board of Trustee of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Brian Kobilka; Daniel Rosenbaum
- Web page: https://patentgrep.com/patent/US20140194595A1

## CPC

- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/70571: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- C12N 9/2462: {Lysozyme (3.2.1.17)}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Certain embodiments provide a method for crystallizing a GPCR. The method may employ a fusion protein comprising: a) a first portion of a G-protein coupled receptor (GPCR), where the first portion comprises the TM1, TM2, TM3, TM4 and TM5 regions of the GPCR; b) a stable, folded protein insertion; and c) a second portion of the GPCR, where the second portion comprises the TM6 and TM7 regions of the GPCR.

## Claims

### Claim 1

A fusion protein comprising, from N-terminus to C-terminus:
a) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR;
b) a stable, folded protein insertion;
c) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR;

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein said GPCR is active.

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein said GPCR is naturally occurring.

### Claim 4 (depends on 1)

The fusion protein of claim 1, wherein said GPCR is non-naturally occurring.

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein said stable, folded protein insertion element is a polypeptide that folds autonomously and is stable in its tertiary folded form.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein said stable, folded protein insertion comprises the amino acid sequence of lysozyme.

### Claim 7 (depends on 1)

A nucleic acid encoding the fusion protein of claim 1.

### Claim 8 (depends on 1)

A cell containing the nucleic acid of claim 1.

### Claim 9 (depends on 8)

The cell of claim 8, wherein said fusion protein is expressed and disposed on the plasma membrane of said cell.

### Claim 10 (depends on 1)

A crystal comprising the fusion protein of claim 1, folded into an active form.

### Claim 11 (depends on 8)

A method comprising:
culturing the cell of claim 8 to produce said fusion protein; and
isolating said fusion protein from said cell.

### Claim 12 (depends on 11)

The method of claim 11, further comprising:
crystallizing said fusion protein to make crystals.

### Claim 13 (depends on 12)

The method of claim 12, wherein said method comprises combining said fusion protein with lipid prior to crystallization.

### Claim 14 (depends on 13)

The method of claim 13, wherein said fusion protein is crystallized using a bicelle crystallization method or a lipidic cubic phase crystallization method.

### Claim 15 (depends on 12)

The method of claim 12, further comprising:
obtaining atomic coordinates of said fusion protein from said crystal.

### Claim 16 (depends on 1)

A method of determining a crystal structure, comprising:
receiving a fusion protein of claim 1,
crystallizing said fusion protein to produce a crystal; and
obtaining atomic coordinates of said fusion protein from said crystal.

### Claim 17 (depends on 1)

A method of determining a crystal structure, comprising:
forwarding a fusion protein of claim 1 to a remote location,
receiving atomic coordinates of said fusion protein.