# US 10,479,815 B2 · Protein purification using intein-hydrophobin tag and alcohol precipitation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,479,815 B2 - Document ID: US10479815B2 - Status: Granted - Kind: B2 - Application number: 16/055,041 - Filed: 2018-08-04 - Published: 2018-12-27 - Granted: 2019-11-19 - Earliest claimed: 2018-08-04 - Applicants: Sareh Arjmand; Sahar Nejatipoor; Seyed Omid Ranaei Siadat - Inventors: Sareh Arjmand; Sahar Nejatipoor; Seyed Omid Ranaei Siadat - Web page: https://patentgrep.com/patent/US10479815B2 ## CPC - C07K 1/30: by precipitation - C07K 1/026: {by fragment condensation in solution} - C07K 16/22: against growth factors {; against growth regulators} - C12N 15/815: {for yeasts other than Saccharomyces} - C07K 2319/01: containing a localisation/targetting motif - C07K 2319/92: containing an intein ("protein splicing")domain - 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) ## Abstract A method of downstream purification of a recombinant protein in fusion with a hydrophobin-intein tag using alcohol precipitation. The method comprises (1) constructing a plasmid expressing a fusion protein in a host cell, wherein the fusion protein includes a target protein domain, an intein, and a hydrophobin domain; (2) culturing the host cell transfected with the plasmid forming a cell culture medium; (3) separating a cell culture supernatant containing the fusion protein from the cell culture medium; and (4) purifying a substantial amount of the target protein from the cell culture supernatant without its hydrophobin-intein fusion tag using alcohol precipitation. ## Claims ### Claim 1 A method of purifying a target protein, the method comprising: (a) preparing a cell culture supernatant comprising a fusion protein, wherein the fusion protein comprising: (i) a target protein domain; (ii) a self-cleaving intein; (iii) at least one hydrophobin protein domain, wherein the intein is located between the target protein domain and the hydrophobin domain; (b) adding a C1-C3 alcohol to the cell culture supernatant making a first alcohol solution; (c) stirring the first alcohol solution forming a first stirred solution; (d) centrifuging the first stirred solution forming a first precipitate and a first supernatant, wherein the first supernatant comprising the fusion protein; (e) separating the first supernatant comprising the fusion protein; (f) adding a C1-C3 alcohol to the first supernatant making the second alcohol solution; (g) stirring the second alcohol solution forming a second stirred solution; (h) centrifuging the second stirred solution forming a second supernatant and a second precipitate, wherein the second precipitate comprising the target protein; and (i) separating the second precipitate comprising the target protein to yield a substantial amount of the purified target protein, wherein the intein is Ssp DnaB mini-intein from Synechocystis, wherein the volume fraction of C1-C3 alcohol added in making the first alcohol solution in step (b) is from about 50% (v/v) to about 75% (v/v), and wherein the volume fraction of the C1-C3 alcohol added in making the second alcohol solution in step (f) is from about 50% (v/v) to about 66.66% (v/v). ### Claim 2 (depends on 1) The method of claim 1, wherein the cell culture supernatant is prepared comprising the steps: (i) constructing a plasmid expressing the fusion protein in a host cell; (ii) culturing the host cell transfected with the plasmid forming a recombinant cell culture medium, wherein the fusion protein is expressed in a plurality of the host cells; (iii) allowing the fusion protein to leave the host cells either by cell secretion or cell lysis into the recombinant cell culture medium; (iv) centrifuging the recombinant cell culture medium forming the cell culture supernatant comprising the fusion protein; and (v) separating the cell culture supernatant to be used for the purification of the target protein. ### Claim 3 (depends on 1) The method of claim 1, wherein the intein undergoes c-terminal cleavage as a result of a shift in pH and temperature. ### Claim 4 (depends on 1) The fusion protein of claim 1, wherein the intein comprises an additional Cystein amino acid at its C-terminal for improving splicing. ### Claim 5 (depends on 1) The fusion protein of claim 1, wherein the at least one hydrophobin domain is covalently attached to the intein by an amino acid linker. ### Claim 6 (depends on 1) The method of claim 1, wherein the target protein is vascular endothelial growth factor A165. ### Claim 7 (depends on 1) The method of claim 1, wherein the target protein is human alpha 1-antitrypsin. ### Claim 8 (depends on 1) The method of claim 1, wherein the hydrophobin is hydrophobin number II from Trichoderma reesei. ### Claim 9 (depends on 1) The method of claim 1, wherein the first supernatant is separated from the first precipitate by decanting the first supernatant. ### Claim 10 (depends on 1) The method of claim 1, wherein the second precipitate is separated from the second supernatant by decanting the second supernatant. ### Claim 11 (depends on 1) The method of claim 1, wherein the volume fraction of C1-C3 alcohol added in making the first alcohol solution in step (b) is about 66.66% (v/v), and the alcohol is C2 alcohol. ### Claim 12 (depends on 1) The method of claim 1, wherein the volume fraction of the C1-C3 alcohol in making the second alcohol solution in step (f) is about 54.54 (v/v), and the alcohol is C2 alcohol. ### Claim 13 (depends on 2) The method of claim 2, wherein the host cell is a strain from Pichia pastoris. ### Claim 14 (depends on 2) The method of claim 2, wherein the cell culture supernatant is separated by decanting the cell culture supernatant.