# US 9,738,692 B2 · Process for production of recombinant proteins as a soluble form > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,738,692 B2 - Document ID: US9738692B2 - Status: Granted - Kind: B2 - Application number: 14/476,624 - Filed: 2014-09-03 - Published: 2015-03-26 - Granted: 2017-08-22 - Earliest claimed: 2006-09-19 - Assignees: Jnc Corporation - Applicants: Jnc Corporation - Inventors: Satoshi Inouye; Yuiko Sahara - Web page: https://patentgrep.com/patent/US9738692B2 ## CPC - C07K 14/31: from Staphylococcus (G) - C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)} - C12N 15/62: DNA sequences coding for fusion proteins - C07K 2319/61: containing an enzyme fusion for detection (lacZ, luciferase) - C07K 2319/705: containing a protein-A fusion - C12Y 113/12005: Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase - C12Y 113/12007: Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase - C12Y 113/12013: Oplophorus-luciferin 2-monooxygenase (1.13.12.13) - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract A target protein is prepared as soluble protein using a recombinant protein expression system. An expression vector is used that includes (1) an expression-inducible promoter sequence; (2) a first coding sequence including a polynucleotide coding for a polypeptide that is represented by the formula (Z)n; and (3) a second coding sequence that includes a polynucleotide that codes for a target protein. A method of producing the target protein is also used that includes expressing protein using this expression vector. ## Claims ### Claim 1 A fusion protein that can be expressed as a soluble protein, comprising: (1) a first amino acid sequence comprising the amino acid sequence of a polypeptide that is represented by the formula (Z)n, wherein n represents an integer from 1 to 5 and Z represents polypeptide selected from the group of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, (b) a polypeptide comprising an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 1, and (c) a polypeptide comprising an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 1, and that has the capacity when expressed as a fusion protein with a target protein to render said fusion protein expressible as a soluble protein; and (2) a second amino acid sequence comprising the amino acid sequence of a target protein, wherein the fusion protein further comprises, on the amino terminal side of the first amino acid sequence, an amino acid sequence that facilitates purification, and; wherein the expression of the fusion protein comprising the amino acid sequence that facilitates purification, an amino acid sequence of the polypeptide that is represented by the formula (Z)n and an amino acid sequence of the target protein is induced in Escherichia coli, and the fusion protein accumulates within the Escherichia coli as a soluble protein so that the fusion protein collected from within the Escherichia coli has higher solubility rate than non-(Z)n fusion protein; and wherein the amino acid sequence that facilitates the purification is a histidine tag sequence; and wherein the target protein is at least one selected from the group of apoaequorin, Gaussia luciferase, and Oplophorus luciferase. ### Claim 2 (depends on 1) The fusion protein according to claim 1, wherein the polypeptide represented by formula (Z)n is a polypeptide represented by (Z)2. ### Claim 3 (depends on 2) The fusion protein according to claim 2, wherein the polypeptide represented by (Z)2 is a polypeptide selected from the group of (d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, (e) a polypeptide that comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 3, and that has the capacity when expressed as a fusion protein with a target protein to render said fusion protein expressible as a soluble protein, and (f) a polypeptide that comprises an amino acid sequence having at least 98% identity with the amino acid sequence of SEQ ID NO: 3, and that has the capacity when expressed as a fusion protein with a target protein to render said fusion protein expressible as a soluble protein. ### Claim 4 (depends on 1) The fusion protein according to claim 1, further comprising, between the first amino acid sequence and the second amino acid sequence, an amino acid sequence comprising the amino acid sequence of a cleavable linker peptide. ### Claim 5 (depends on 4) The fusion protein according to claim 4, wherein the cleavable linker peptide is a linker peptide having a protease cleavage site. ### Claim 6 A fusion protein that can be expressed as a soluble protein and that is represented by the formula (Z)n-L-X, wherein n represents an integer from 1 to 5; L represents a cleavable linker peptide; Z represents polypeptide selected from the group of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, (b) a polypeptide comprising an amino acid sequence that has at least 95% identity with the amino acid sequence of SEQ ID NO: 1, and (c) a polypeptide comprising an amino acid sequence that has at least 98% identity with the amino acid sequence of SEQ ID NO: 1, and X represents the amino acid sequence of a target protein, wherein the fusion protein further comprises, on the amino terminal side of Z, an amino acid sequence that facilitates purification, and; wherein the expression of the fusion protein is induced in Escherichia coli, and the fusion protein accumulates within the Escherichia coli as a soluble protein so that the fusion protein collected from within the Escherichia coli has higher solubility rate than non-(Z)n fusion protein; and wherein the amino acid sequence that facilitates the purification is a histidine tag sequence; and wherein the target protein is at least one selected from the group of apoaequorin, Gaussia luciferase, and Oplophorus luciferase.