# US 10,501,502 B2 · Allophycocyanin alpha-subunit evolved labeling proteins (smURFPs)

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- Number: US 10,501,502 B2
- Document ID: US10501502B2
- Status: Granted
- Kind: B2
- Application number: 15/821,574
- Filed: 2017-11-22
- Published: 2018-07-19
- Granted: 2019-12-10
- Earliest claimed: 2016-11-22
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California; Wendy M. G. Tsien
- Inventors: Erik Rodriguez; John Lin; Roger Tsien; Richard Ting; Geraldine Tran
- Web page: https://patentgrep.com/patent/US10501502B2

## CPC

- C07K 14/195: from bacteria
- 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)

## Abstract

The invention provides evolved red-shifted smURFPs. In some embodiments, the smURFPs are characterized by a) increased Fluorescence of smURFP as compared to infrared FPs IFP1.4 and iRFP713; b) expressing efficiently with minimal toxicity; c) does not require a lyase to covalently attach its chromophore, wherein the chromophore is biliverdin; d) exhibit a wavelengths longer than attainable with jellyfish- or coral-derived FPs using smURFP and IFP2.0; e) allows for functional fusion to hCdt1(30/120) as compared to jellyfish- or coral-derived FPs mAG, eGFP, and mRFP1 which are nonfunctional; and f) allows for deep tissue imaging.

## Claims

### Claim 1

A small ultra-red fluorescent protein (smURFP) variant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:13, and SEQ ID NO:13 further comprising C52A and A96C mutations.

### Claim 2 (depends on 1)

The smURFP variant polypeptide according to claim 1, wherein said smURFP variant polypeptide is a dimer comprising two smURFP variant polypeptides conjugated by a linker.

### Claim 3 (depends on 2)

The smURFP variant polypeptide according to claim 2, wherein said linker is an amino-acid linker.

### Claim 4 (depends on 3)

The smURFP variant polypeptide according to claim 3, wherein said amino-acid linker comprises 23 amino acids.

### Claim 5 (depends on 1)

The smURFP variant polypeptide according to claim 1, wherein said smURFP variant polypeptide is a dimer comprising one smURFP conjugated to a second fluorescent protein by a linker.

### Claim 6 (depends on 1)

The smURFP variant polypeptide according to claim 1, wherein said smURFP variant polypeptide is a dimer comprising one smURFP conjugated to a second smURFP.

### Claim 7 (depends on 5)

The smURFP variant polypeptide according to claim 5, wherein said second fluorescent protein is selected from the group consisting of eGFP, mCherry, mCardinal, IFP1.4, IFP2.0, and iRFP713.

### Claim 8 (depends on 1)

A nucleic acid encoding the smURFP variant polypeptide of claim 1.

### Claim 9 (depends on 8)

An expression vector comprising the nucleic acid of claim 8.

### Claim 10 (depends on 9)

An isolated host cell comprising the expression vector of claim 9.

### Claim 11 (depends on 10)

A method of producing a smURFP comprising: a) culturing a host cell according to claim 10 under conditions wherein said polypeptide is produced; and b) purifying said polypeptide.

### Claim 12 (depends on 1)

A biosensor comprising a smURFP according to claim 1.