# US 7,608,415 B2 · Analysis of intracellular modifications

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- Number: US 7,608,415 B2
- Document ID: US7608415B2
- Status: Granted
- Kind: B2
- Application number: 11/170,123
- Filed: 2005-06-29
- Published: 2006-01-26
- Granted: 2009-10-27
- Earliest claimed: 2004-06-30
- Assignees: DiscoveRx Corporation
- Inventors: Joseph Horecka; Peter Fung; Richard M. Eglen
- Web page: https://patentgrep.com/patent/US7608415B2

## CPC

- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- C12Q 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
- G01N 33/5002: {Partitioning blood components}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/5023: {on expression patterns}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 33/573: for enzymes or isoenzymes
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- G01N 2333/924: acting on glycosyl compounds (3.2)
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Improved methods of determining the intracellular state of a protein as well as modifications of the protein are provided by introducing a surrogate fusion protein comprising a member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with the surrogate fusion protein to a change in environment, e.g. a candidate drug, the cells are lysed, the lysate separated into fractions or bands, conveniently by gel electrophoresis and transferring the proteins by Western blot to a membrane. The bands on the membrane are developed using the other member of the enzyme fragment complementation complex and a substrate providing a detectable signal. The method is found to provide high sensitivity and the ability to observe modifications of the target protein.

## Claims

### Claim 1

A method for performing a Western blot for determining modification of an intracellular target protein in a transfected cell using a fusion protein serving as a surrogate protein for said-target protein, said surrogate protein comprising an enzyme donor (ED), wherein said ED is a member of an enzyme fragment complementation pair of β-galactosidase that forms an active enzyme with the complementary enzyme acceptor fragment (EA) and said surrogate protein is modified in said cell in the same manner as said target protein, said modification occurring as a result of changes in the environment of said cell where said modifications result in a change in the migration rate of said surrogate protein in gel electrophoresis, wherein said method comprises:
lysing said transfected cell, transformed with an expression construct of said fusion protein resulting in intracellular expression of said fusion protein, to form a lysate;
separating said lysate by gel electrophoresis into bands where said fusion protein and intracellular modifications thereof comprising said ED are separated by at least one of size and mass-to-charge ratio;
transferring said bands to a membrane;
adding EA and substrate to said membrane to visualize modified and unmodified fusion protein bands free of other bands; and
determining from said bands whether said target protein is modified as a result of said changes in the environment of said cell.

### Claim 2 (depends on 1)

A method according to claim 1, wherein said ED is less than about 100 amino acids.

### Claim 3 (depends on 1)

A method according to claim 1, wherein said ED is at the C-terminus of said target protein.

### Claim 4 (depends on 1)

A method according to claim 1, further comprising, prior to lysing said cell, further comprising the step of contacting said cell with an agent to determine the effect of said agent on the state of said target protein in said cell.

### Claim 5 (depends on 4)

A method according to claim 4, wherein said agent-acts to provide RNA interference (RNAi) for a protein other than said target protein.

### Claim 6 (depends on 1)

A method according to claim 1, including the additional step of determining a natural target protein on said membrane.

### Claim 7 (depends on 1)

A method according to claim 1, wherein said Western blot employs a polyvinyldifluoride (PVDF) membrane.

### Claim 8 (depends on 1)

A method according to claim 1, wherein said Western blot employs a nitrocellulose membrane.

### Claim 9 (depends on 4)

A method according to claim 4, wherein said agent is a candidate drug.

### Claim 10 (depends on 1)

A method according to claim 1, wherein said changes are selected from the group consisting of degradation, phosphorylation, glycosylation or acylation.