# US 8,541,175 B2 · Detection of molecular interactions using a reduced affinity enzyme complementation reporter system

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- Number: US 8,541,175 B2
- Document ID: US8541175B2
- Status: Granted
- Kind: B2
- Application number: 11/717,579
- Filed: 2007-03-12
- Published: 2007-11-29
- Granted: 2013-09-24
- Earliest claimed: 2006-03-13
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: Tom Wehrman; Helen M. Blau
- Inventors: Tom Wehrman; Helen M. Blau
- Web page: https://patentgrep.com/patent/US8541175B2

## CPC

- C12Q 1/34: involving hydrolase
- C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection}
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Methods and compositions for detecting molecular interactions are provided. Aspects of the invention include the use of a reduced affinity enzyme complementation reporter system. Also provided are systems and kits for use in practicing embodiments of the methods.

## Claims

### Claim 1

A method of determining whether a first and second protein interact by binding to each other, said method comprising:
(a) providing a mammalian cell comprising:
(i) a first fusion protein of said first protein and a first β-galactosidase fragment of a full length β-galactosidase enzyme, wherein said first β-galactosidase fragment is a variant minimal N-terminal β-galactosidase peptide about 60 amino acids or less in length; and
(ii) a second fusion protein of said second protein and a second β-galactosidase fragment including greater than 60% of the full-length β-galactosidase enzyme;
wherein said first and second β-galactosidase fragments have an affinity for each other which provides a known level of β-galactosidase activity in the absence of an interaction between said first and second proteins that is lower than the activity observed in the presence of an interaction between said first and second proteins, wherein said variant minimal N-terminal β-galactosidase peptide comprises a point mutation, insertion or deletion between amino acid residues 29 to 41, or a truncation terminating between amino acid residues 48 and 43, as set forth in a β-galactosidase peptide sequence according to SEQ ID NO: 7; and
(b) evaluating said cell for β-galactosidase activity to determine whether said first and second proteins interact by said binding to each other, thereby complementing said first β-galactosidase fragment and said second β-galactosidase fragment.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said providing comprises introducing nucleic acids encoding said first and second fusion proteins into said cell.

### Claim 3 (depends on 2)

The method according to claim 2, wherein said nucleic acids are introduced into said cell sequentially.

### Claim 4 (depends on 2)

The method according to claim 2, wherein said nucleic acids are introduced into said cell simultaneously.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said method further comprises contacting said cell with a candidate interaction modulatory agent prior to said evaluating.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said evaluating comprises comparing observed β-galactosidase activity to said known level of β-galactosidase activity.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said variant minimal N-terminal β-galactosidase peptide comprises a point mutation between amino acids 29 to 41 that is a substitution.

### Claim 8 (depends on 1)

The method according to claim 1, wherein said variant minimal N-terminal β-galactosidase peptide comprises a deletion between amino acids 29 to 41.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said point mutation, insertion or deletion occurs between residues 31 and 41.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said interaction occurs at an intracellular location.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said interaction occurs at a plasma-membrane location.

### Claim 12 (depends on 1)

The method according to claim 1 wherein said variant minimal N-terminal β-galactosidase peptide has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

### Claim 13 (depends on 1)

The method according to claim 1 wherein the first protein and second protein are in a membrane of said cell.