Genetically encoded photocleavable proteins

Patent metadata

Field	Value
Publication number	US-2017190749-A1
Application number	US-201515116168-A
Country	US
Kind code	A1
Filing date	Feb 10, 2015
Priority date	Feb 11, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

Genetically encoded, photocleavable proteins are derived from a fluorescent protein. Upon illumination, the proteins photocleave and spontaneously dissociate into two or more fragments or release one end of an internal loop.

First claim

1 . A photocleavable genetically encoded protein comprising a His-Tyr-Gly chromophore, wherein the protein spontaneously dissociates into at least two fragments, or releases one end of a loop insertion, following photocleavage.

2 . The photocleavable protein of claim 1 which changes from green to red fluorescence to a non-fluorescent state upon photocleavage, and which has a structure comprising an α-helix comprising the chromophore His-Tyr-Gly within β-barrel comprising a β-sheet, the protein comprising a sequence break between the α-helix and a β-sheet, the sequence break consisting of a C-terminus and a N-terminus or a loop insertion comprising a functional polypeptide sequence.

3 . The photocleavable protein of claim 1 or 2 which is a circular permutation of mMaple.

4 . The photocleavable protein of claim 2 or 3 wherein the sequence break is within 12 amino acid residues of the His-Tyr-Gly chromophore.

5 . The photocleavable protein of claim 3 wherein the sequence break comprises a loop insertion comprising a functional polypeptide sequence.

6 . The photocleavable protein of claim 3 wherein the sequence break is positioned between the E78 and D79 residues of mMaple.

7 . The photocleavable protein of claim 6 comprising mutations E78R and D79V.

8 . The photocleavable protein of claim 7 further comprising mutations M64T and I15N.

9 . The photocleavable protein of claim 8 further comprising a F177Y mutation.

10 . The photocleavable protein of claim 9 further comprising one or more of the following mutations: S172G, E116G, V146A, V153E, A187P.

11 . The photocleavable protein of claim 1 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16, or a substantially similar amino acid sequence wherein the protein is photocleavable and dissociates into at least one fragment, or releases one end of a loop insertion.

12 . The photocleavable protein of claim 11 comprising one or both sequences of position 1 to position 82 and position 83 to position 237 in SEQ ID NO: 16.

13 . A nucleic acid encoding the photocleavable protein of any one of claims 1 to 13 , or comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15.

14 . The nucleic acid of claim 13 , wherein the encoded polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16.

15 . The nucleic acid of claim 14 which is compatible with mammalian or human codon usage.

16 . A recombinant expression vector comprising a nucleic acid of one of claims 13 - 15 , operably linked with transcriptional and translational regulatory regions or sequences to provide for expression of the nucleic acid in a host cell.

17 . An isolated host cell comprising the vector of claim 16 .

18 . A transgenic animal comprising the nucleic acid of one of claims 13 - 15 .

19 . (canceled)

20 . (canceled)

21 . (canceled)

22 . A method of localizing a protein within a cell comprising the step of providing a photocleavable genetically encoded protein comprising a His-Tyr-Gly chromophore, wherein said protein spontaneously dissociates into at least two fragments following photocleavage, wherein one or more fragments comprises a localization tag or an exclusion tag, and photocleaving the protein.

23 . The method of claim 22 adapted to modulate gene expression, wherein a dissociated fragment comprises a nuclear localization tag and a transcription factor.

24 . A method of enzyme activation comprising the step of providing a photocleavable genetically encoded protein construct comprising a His-Tyr-Gly chromophore and the enzyme and an enzyme inhibitor, wherein said protein construct spontaneously dissociates into at least two fragments following photocleavage, wherein a first fragment comprises the enzyme, and a second fragment comprises the inhibitor.

25 . (canceled)

26 . (canceled)

27 . A method of purifying a protein of interest using a purification substrate having an affinity tag, comprising the step of providing a genetically encoded photocleavable protein comprising a His-Tyr-Gly chromophore and a sequence break consisting of a C-terminus and a N-terminus, wherein the protein spontaneously dissociates into two fragments following photocleavage, wherein one fragment comprises an affinity tag which specifically binds to the substrate affinity tag, and the other fragment comprises the protein of interest.

Assignees

• Univ Alberta

Inventors

• Campbell Robert
• Zhang Wei
• Hoi Hiofan

Classifications

• A01K67/0275

  Genetically modified vertebrates, e.g. transgenic · CPC title

• C07K14/43595 Primary

  from coelenteratae, e.g. medusae · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• C12N15/635

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title

• C07K1/107

  by chemical modification of precursor peptides · CPC title