# US 10,308,969 B2 · Method for preparing polymeric protein composed of monomeric protein produced by fusing protein having immunoglobulin fold structure to protein capable of serving as subunit structure > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,969 B2 - Document ID: US10308969B2 - Status: Granted - Kind: B2 - Application number: 14/104,139 - Filed: 2013-12-12 - Published: 2014-04-10 - Granted: 2019-06-04 - Earliest claimed: 2011-06-23 - Assignees: Ajinomoto Co., Inc. - Applicants: Ajinomoto Co., Inc. - Inventors: Daisuke Ejima; Haruna Sato; Kouhei Tsumoto; Masayo Date - Web page: https://patentgrep.com/patent/US10308969B2 ## CPC - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C07K 1/1136: {by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents} - C07K 14/36: from Actinomyces; from Streptomyces (G) - C07K 16/40: against enzymes - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/00: Fusion polypeptide - 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 invention provides a method for producing a multimeric protein composed of a monomeric protein, wherein the monomeric protein is obtained by fusing a protein having an immunoglobulin fold structure to a protein that can serve as a subunit structure, the method including the steps of: (A) preparing the monomeric protein having an insoluble granular form in cells of a microorganism; (B) solubilizing the monomeric protein prepared in step (A) with an aqueous solution containing lauroyl-L-Glu; (C) diluting a solution obtained in step (B) in a buffer containing arginine hydrochloride to lower a concentration of lauroyl-L-Glu; and (D) replacing a solvent of a solution obtained in step (C) with a buffer using gel filtration chromatography or the like. ## Claims ### Claim 1 A method for producing a multimeric protein consisting of two or more monomeric protein, wherein said monomeric protein is obtained by fusing a protein having an immunoglobulin fold structure to either a protein or a functional fragment thereof that can serve as a subunit structure, the method comprising the steps of: (A) preparing a monomeric protein having an insoluble granular form in cells of a microorganism, the monomeric protein obtained by fusing a first protein having an immunoglobulin fold structure to a second different protein that can serve as a subunit structure; (B) solubilizing the monomeric protein prepared in step (A) with an aqueous solution comprising lauroyl glutamic acid and/or a salt thereof, resulting in a first solution; (C) adding a phosphate buffer to the first solution obtained in step (B) so that the concentration of the lauroyl glutamic acid and/or salt thereof is 2 to 5%; (D) diluting the solution obtained in step (C) in a buffer comprising arginine and/or an arginine derivative to lower the concentration of lauroyl glutamic acid and/or a salt thereof, resulting in a second solution; and (E) replacing a solvent of the second solution obtained in step (C) with a buffer, resulting in removal said lauroyl glutamic acid and/or a salt thereof, using a method selected from the group consisting of: (e1) column chromatography selected from the group consisting of gel filtration chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and combination thereof; (e2) ultrafiltration; (e3) dialysis; and (e4) combinations thereof. ### Claim 2 (depends on 1) The method according to claim 1, wherein the monomeric protein is a fusion protein of scFv and streptavidin. ### Claim 3 (depends on 2) The method according to claim 2, wherein the scFv is selected from the group consisting of HyHEL-10 scFv and D1.3 scFv. ### Claim 4 (depends on 2) The method according to claim 2, wherein the streptavidin is full length streptavidin or a functional fragment thereof. ### Claim 5 (depends on 1) The method according to claim 1, wherein the arginine derivative is selected from the group consisting of arginine having an acyl group with 1 to 6 carbon atoms, arginine butyl ester, agmatine, and argininic acid. ### Claim 6 (depends on 1) The method according to claim 1, wherein the arginine is arginine hydrochloride.