Genetically encoded photocleavable proteins

Patent metadata

Field	Value
Publication number	US-10370420-B2
Application number	US-201515116168-A
Country	US
Kind code	B2
Filing date	Feb 10, 2015
Priority date	Feb 11, 2014
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

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

What is claimed is:

1. A photocleavable protein comprising a His-Tyr-Gly chromophore, which protein comprises a circular permutation of the amino acid sequence of SEQ ID. NO. 17, 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 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and is photocleavable and dissociates into at least two fragments, or releases one end of a loop insertion.

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

3. A recombinant expression vector comprising a nucleic acid of claim 2 , operably linked with transcriptional and translational regulatory regions or sequences to provide for expression of the nucleic acid in a host cell.

4. A host cell comprising the vector of claim 3 .

5. A method of localizing a protein within a cell comprising the step of providing a photocleavable genetically encoded protein of claim 1 , wherein one or more fragments comprises a localization tag or an exclusion tag, and photocleaving the protein.

6. The method of claim 5 adapted to modulate gene expression, wherein a dissociated fragment comprises a nuclear localization tag and a transcription factor.

7. A method of enzyme activation comprising the step of providing a photocleavable genetically encoded protein construct comprising protein of claim 1 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.

8. 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 of claim 1 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

• C12N15/635

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

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• A01K67/0275

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

• G01N33/6803

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

• C12N9/00

  Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00) · CPC title