# US 8,628,939 B2 · Expression vector containing the major envelope protein P9 of cystovirus PHI6 as a fusion partner, and process for producing a membrane protein using the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,628,939 B2 - Document ID: US8628939B2 - Status: Granted - Kind: B2 - Application number: 13/058,425 - Filed: 2009-08-13 - Published: 2011-11-24 - Granted: 2014-01-14 - Earliest claimed: 2008-08-13 - Assignees: Foundation of Soongsil University-Industry Cooperation - Applicants: Dong-bin Lim; Yuna Jung - Inventors: Dong-bin Lim; Yuna Jung - Web page: https://patentgrep.com/patent/US8628939B2 ## CPC - C07K 14/005: from viruses - C12N 15/62: DNA sequences coding for fusion proteins - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C07K 2319/00: Fusion polypeptide - C07K 2319/035: containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins - C07K 2319/50: containing protease site - C12N 2795/12022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - 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) - 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)} ## Abstract The present invention relates to a membrane protein expression vector containing the major envelope protein P9 of Cystovirus phi6 as a fusion partner, to cells transformed by the expression vector, and to a process for producing membrane proteins using the cells. Target proteins can be effectively expressed by the expression vector of the present invention. ## Claims ### Claim 1 An expression vector comprising a major envelope protein P9 gene of Cystovirus phi6, a multicloning site (MCS) for inserting a target membrane protein, and a protease recognition site located between a P9 gene and the MCS. ### Claim 2 (depends on 1) The expression vector of claim 1, wherein said major envelope protein P9 has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. ### Claim 3 (depends on 1) The expression vector of claim 1, wherein said major envelope protein P9 has the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. ### Claim 4 (depends on 1) The expression vector of claim 1, wherein said expression vector comprises a promoter, a protein P9 gene, a protease recognition site, an MCS and a histidine tag which are linked in a 5′- to 3′-direction in the form of being fused to a translational fusion. ### Claim 5 (depends on 4) The expression vector of claim 4, wherein said expression vector has the nucleotide sequence of SEQ ID NO: 5. ### Claim 6 (depends on 4) The expression vector of claim 4, wherein said expression vector additionally comprises an extra transmembrane (TM) domain between said protein P9 gene and said protease recognition site. ### Claim 7 (depends on 6) The expression vector of claim 6, wherein said extra TM domain is derived from a TM domain-containing protein selected from the group consisting of a protein P9 of Cystovirus, a protein P10 of Cystovirus, a major coat protein of Pseudomonas phage Pf3 and a major coat protein of Bacteriophage M13. ### Claim 8 (depends on 6) The expression vector of claim 6, wherein said expression vector comprises a base sequence which encodes a protein of SEQ ID NO: 37, 39 or 41. ### Claim 9 (depends on 8) The expression vector of claim 8, wherein said a nucleotide sequence is selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40. ### Claim 10 (depends on 4) The expression vector of claim 4, wherein said promoter is selected from the group consisting of T7 promoter, T5 promoter and tac promoter. ### Claim 11 (depends on 4) The expression vector of claim 4, wherein said protease is selected from the group consisting of thrombin, Tev and enterokinase. ### Claim 12 (depends on 1) The expression vector of claim 1, wherein said expression vector is pRphi6-9 (Depository number: KCTC 11373BP). ### Claim 13 (depends on 1) A cell transformed by the expression vector in claim 1. ### Claim 14 (depends on 13) The cell of claim 13, wherein said cell is a cell of a microorganism or an animal. ### Claim 15 (depends on 13) The cell of claim 13, wherein said cell is Escherichia coli EPI300/pRphi6-9 (Depository number: KCTC 11373BP). ### Claim 16 (depends on 1) A process for producing a target membrane protein comprising inserting a gene encoding the target membrane protein into the MCS of the expression vector of claim 1, transforming said gene into a cell, and culturing the transformed cell. ### Claim 17 (depends on 16) The process of claim 16, wherein said membrane protein is selected from the group consisting of a membrane receptor, an ion channel, a membrane transporter, a pump, a membrane enzyme, a ligand and a receptor for intercellular communication, a linker for linking cells, a membrane vesicle for intracellular material transport, a ligand and a receptor of endo- and exo-cytosis, a biomembrane protein relating to a viral life cycle, an antibody or a part thereof, and a toxoprotein.