# US 10,865,394 B2 · Toolkit for the production of post-translationally modified proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,394 B2 - Document ID: US10865394B2 - Status: Granted - Kind: B2 - Application number: 15/710,451 - Filed: 2017-09-20 - Published: 2018-01-11 - Granted: 2020-12-15 - Earliest claimed: 2015-07-27 - Assignees: Washington University - Applicants: Kristen Naegle; Roman Sloutsky - Inventors: Kristen Naegle; Roman Sloutsky - Web page: https://patentgrep.com/patent/US10865394B2 ## CPC - C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7) - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C07K 2319/00: Fusion polypeptide - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - 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 present disclosure provides a system to produce soluble, folded, and post-translationally modified proteins. The system includes a fusion protein comprising a catalytic domain of an enzyme involved in post-translational protein modification and a targeting domain, and a substrate protein comprising a protein of interest and a sequence that interacts with the targeting domain. The present disclosure also provides polynucleotide sequences encoding fusion proteins and substrate proteins, vectors for expressing polynucleotide sequences, vectors comprising the polynucleotide sequences, and isolated cells comprising said vectors. ## Claims ### Claim 1 A method of phosphorylating a substrate protein of interest comprising contacting a fusion protein and a substrate protein for a period of time sufficient to allow for phosphorylation of the substrate protein by the fusion protein to occur, wherein (a) the fusion protein comprises a kinase catalytic domain, a targeting domain and a first linker, wherein the targeting domain is a SH3 (src Homology 3) domain and the kinase catalytic domain and the targeting domain are joined by the first linker; and (b) the substrate protein comprises a polypeptide of interest, a sequence that interacts with the targeting domain of the fusion protein, a protease cleavage site, and an optional second linker; wherein (i) the sequence that interacts with the targeting domain is a polyproline sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, a variant with at least 80% sequence identity to SEQ ID NO: 2, 3 or 4 that is capable of binding to an SH3 domain and a sequence of about 5 to about 20 amino acids in length and contains the sequence proline-Xaa-Xaa-proline, wherein Xaa is any amino acid; (ii) the protease cleavage site is between the polypeptide of interest and the second linker when the second linker is present, and between the polypeptide of interest and the sequence that interacts with the targeting domain when the second linker is not present; and (iii) wherein the first linker contains repeats of two, oppositely-charged, amino acids, and the first linker is about 10 to about 30 amino acids in length and/or the second linker contains repeats of two, oppositely-charged, amino acids, and the second linker is about 10 to about 30 amino acids in length. ### Claim 2 (depends on 1) The method of claim 1, wherein the oppositely-charged amino acids of the first linker and/or the second linker are lysine and glutamic acid. ### Claim 3 (depends on 1) The method of claim 1, wherein the fusion protein and/or the substrate protein further comprises at least one N-terminal or C-terminal peptide tag selected from the group consisting of an affinity tag, a purification tag, a solubility tag, and a stability tag. ### Claim 4 (depends on 3) The method of claim 3, wherein the fusion protein and/or the substrate protein further comprises a second protease cleavage site, wherein the second protease cleavage site is positioned relative to the peptide tag such that cleavage at the second protease site results in removal of the protein tag from the protein. ### Claim 5 (depends on 1) The method of claim 1, wherein the fusion protein and/or the substrate protein comprises (a) at least one affinity tag, purification tag, or tag that is a combination thereof; and (b) at least one solubility tag or stability tag. ### Claim 6 (depends on 5) The method of claim 5, wherein the fusion protein and/or the substrate protein comprises (a) at the C-terminus, at least one affinity tag, purification tag, or tag that is a combination thereof, and at the N-terminus, at least one stability tag or solubility tag; or (b) at the N-terminus, at least one affinity tag, purification tag, or tag that is a combination thereof, and at the C-terminus, at least one stability tag or solubility tag. ### Claim 7 (depends on 5) The method of claim 5, wherein the fusion protein further comprises a protease cleavage site, and the protease cleavage site is positioned relative to (a) the at least one affinity tag, purification tag, or tag that is a combination thereof, and/or (b) the at least one solubility tag or stability tag such that cleavage at the protease site results in removal of the tag from the protein. ### Claim 8 (depends on 1) The method of claim 1, wherein the sequence that interacts with the targeting domain is on the N-terminal side of the protease cleavage site. ### Claim 9 (depends on 1) The method of claim 1, wherein the sequence that interacts with the targeting domain is on the C-terminal side of the protease cleavage site. ### Claim 10 (depends on 1) The method of claim 1, wherein the method further comprises purifying the phosphorylated substrate protein.