Allophycocyanin alpha-subunit evolved labeling proteins (smURFPs)

Patent metadata

Field	Value
Publication number	US-10501502-B2
Application number	US-201715821574-A
Country	US
Kind code	B2
Filing date	Nov 22, 2017
Priority date	Nov 22, 2016
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

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.

First claim

What is claimed:

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.

2. 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.

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

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

5. 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.

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

7. 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.

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

9. An expression vector comprising the nucleic acid of claim 8 .

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

11. 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.

12. A biosensor comprising a smURFP according to claim 1 .

Assignees

• Univ California
• Tsien Wendy M G

Inventors

• Rodriguez Erik
• Lin John
• Tsien Roger
• Ting Richard
• Tran Geraldine

Classifications

• C07K14/195 Primary

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