# US 8,853,374 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 8,853,374 B2 - Document ID: US8853374B2 - Status: Granted - Kind: B2 - Application number: 11/902,030 - Filed: 2007-09-18 - Published: 2008-03-27 - Granted: 2014-10-07 - Earliest claimed: 2006-09-19 - Assignees: Jnc Corporation - Applicants: Satoshi Inouye; Yuiko Sahara - Inventors: Satoshi Inouye; Yuiko Sahara - Web page: https://patentgrep.com/patent/US8853374B2 ## 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 An expression vector for expression of a fusion protein, comprising: (1) an expression-inducible promoter sequence, wherein the expression-inducible promoter sequence is a cold-shock promoter sequence selected from the group consisting of a cspA promoter sequence, a cspB promoter sequence, a cspG promoter sequence, a cspl promoter sequence, and a csdA promoter sequence; (2) a first coding sequence comprising a polynucleotide encoding 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 (3) a second coding sequence that comprises a polynucleotide that encodes a target protein, wherein the vector further comprises, on the 5′ side of the first coding sequence, a coding sequence that comprises a polynucleotide that encodes an amino acid sequence that facilitates purification, and wherein the expression of the fusion protein comprising the amino acid sequence that facilitates purification, the amino acid sequence of the polypeptide that is represented by the formula (Z)n and the 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. ### Claim 2 (depends on 1) The vector 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 vector 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 vector according to claim 1, further comprising, between the first coding sequence and the second coding sequence, a coding sequence comprising a polynucleotide encoding a cleavable linker peptide. ### Claim 5 (depends on 4) The vector according to claim 4, wherein the cleavable linker peptide is a linker peptide having a protease cleavage site. ### Claim 6 (depends on 1) The vector according to claim 1, wherein the amino acid sequence that facilitates the purification is a histidine tag sequence. ### Claim 7 (depends on 1) The expression vector according to claim 1, wherein the target protein is an apoprotein of a holoprotein. ### Claim 8 (depends on 1) The expression vector according to claim 1, wherein the target protein is Gaussia luciferase (hGL) or 19 kDa protein that is the catalytic unit of Oplophorus (shrimp) luciferase (KAZ). ### Claim 9 (depends on 1) The expression vector according to claim 1, wherein a temperature for the expression induction is 10 to 20° C. ### Claim 10 An expression vector for expression of a fusion protein, comprising: (1) an expression-inducible promoter sequence, wherein the expression-inducible promoter sequence is a cold-shock promoter sequence selected from the group consisting of a cspA promoter sequence, a cspB promoter sequence, a cspG promoter sequence, a cspl promoter sequence, and a csdA promoter sequence; (2) a first coding sequence comprising a polynucleotide encoding 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 (3) at least one restriction enzyme site that enables the insertion of a second coding sequence that comprises a polynucleotide that encodes a target protein, wherein the vector further comprises, on the 5′ side of the first coding sequence, a coding sequence that comprises a polynucleotide that encodes an amino acid sequence that facilitates purification, and wherein the expression of the fusion protein comprising the amino acid sequence that facilitates purification, the amino acid sequence of the polypeptide that is represented by the formula (Z)n and the 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. ### Claim 11 (depends on 10) The vector according to claim 10, wherein the polypeptide represented by formula (Z)n is a polypeptide represented by (Z)2. ### Claim 12 (depends on 11) The vector according to claim 11, 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 13 (depends on 10) The vector according to claim 10, further comprising, between the first coding sequence and the second coding sequence, a coding sequence comprising a polynucleotide encoding a cleavable linker peptide. ### Claim 14 (depends on 13) The vector according to claim 13, wherein the cleavable linker peptide is a linker peptide having a protease cleavage site. ### Claim 15 (depends on 10) The vector according to claim 10, wherein the amino acid sequence that facilitates the purification is a histidine tag sequence. ### Claim 16 (depends on 10) The expression vector according to claim 10, wherein the target protein is an apoprotein of a holoprotein. ### Claim 17 (depends on 10) The expression vector according to claim 10, wherein the target protein is Gaussia luciferase (hGL) or 19 kDa protein that is the catalytic unit of Oplophorus (shrimp) luciferase (KAZ). ### Claim 18 (depends on 10) The expression vector according to claim 10, wherein a temperature for the expression induction is 10 to 20° C. ### Claim 19 A polynucleotide for expression of a fusion protein comprising an expression-inducible promoter sequence, wherein the expression-inducible promoter sequence is a cold-shock promoter sequence selected from the group consisting of a cspA promoter sequence, a cspB promoter sequence, a cspG promoter sequence, a cspl promoter sequence, and a csdA promoter sequence, and a polynucleotide that encodes the 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 polynucleotide further comprises, on the 5′ side of the first coding sequence, a coding sequence that comprises a polynucleotide that encodes an amino acid sequence that facilitates purification, wherein an expression for the polynucleotide which encodes the fusion protein comprising the amino acid sequence that facilitates purification, the amino acid sequence of the polypeptide that is represented by the formula (Z)n and the 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. ### Claim 20 (depends on 19) The polynucleotide according to claim 19, that is DNA. ### Claim 21 (depends on 19) The polynucleotide according to claim 19, wherein the target protein is an apoprotein of a holoprotein. ### Claim 22 (depends on 19) The polynucleotide according to claim 19, wherein the target protein is Gaussia luciferase (hGL) or 19 kKa protein that is the catalytic unit of Oplophorus (shrimp) luciferase (KAZ). ### Claim 23 (depends on 19) The polynucleotide according to claim 19, wherein a temperature for the expression induction is 10 to 20° C. ### Claim 24 (depends on 1) A method of producing a target protein as a soluble protein, wherein the method comprises: expressing a fusion protein by providing the expression vector according to claim 1. ### Claim 25 (depends on 24) The method according to claim 24, wherein the polypeptide represented by formula (Z)n is a polypeptide represented by (Z)2. ### Claim 26 (depends on 25) The method according to claim 25, wherein the polypeptide represented by the formula (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 27 (depends on 24) The method according to claim 24, further comprising, between the first coding sequence and the second coding sequence, a coding sequence comprising a polynucleotide coding for a cleavable linker peptide. ### Claim 28 (depends on 27) The method according to claim 27, wherein the cleavable linker peptide is a linker peptide having a protease cleavage site. ### Claim 29 (depends on 24) The method according to claim 24, wherein the amino acid sequence that facilitates the purification is a histidine tag sequence.