# US 10,370,420 B2 · Genetically encoded photocleavable proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,420 B2 - Document ID: US10370420B2 - Status: Granted - Kind: B2 - Application number: 15/116,168 - Filed: 2015-02-10 - Published: 2017-07-06 - Granted: 2019-08-06 - Earliest claimed: 2014-02-11 - Applicants: The Governors of the University of Alberta - Inventors: Robert Campbell; Wei Zhang; Hiofan Hoi - Web page: https://patentgrep.com/patent/US10370420B2 ## CPC - C07K 14/43595: {from coelenteratae, e.g. medusae} - A01K 67/0275: Genetically modified vertebrates, e.g. transgenic - C07K 1/107: by chemical modification of precursor peptides - C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes - C12N 9/00: 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) - C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline} - G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins} - 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) - A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS - 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 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 2) 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. ### Claim 4 (depends on 3) A host cell comprising the vector of claim 3. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 5) The method of claim 5 adapted to modulate gene expression, wherein a dissociated fragment comprises a nuclear localization tag and a transcription factor. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 1) 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.