# US 7,314,925 B2 · Constructs and methods for expression of recombinant HCV envelope proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,314,925 B2 - Document ID: US7314925B2 - Status: Granted - Kind: B2 - Application number: 10/128,587 - Filed: 2002-04-24 - Published: 2003-08-14 - Granted: 2008-01-01 - Earliest claimed: 2001-04-24 - Assignees: Innogenetics N.V. - Inventors: Erwin Sablon; Annie Van Broekhoven; Fons Bosman; Erik Depla; Geert Deschamps - Web page: https://patentgrep.com/patent/US7314925B2 ## CPC - C07K 14/005: from viruses - A61P 31/14: for RNA viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - A61K 2039/5258: {Virus-like particles} - C07K 2319/02: containing a signal sequence - C07K 2319/50: containing protease site - C12N 2770/24222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2770/24223: Virus like particles [VLP] - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The current invention relates to vectors and methods for efficient expression of HCV envelope proteins in eukaryotic cells. More particularly said vectors comprise the coding sequence for an avian lysozyme signal peptide or a functional equivalent thereof joined to a HCV envelope protein or a part thereof. Said avian lysozyme signal peptide is efficiently removed when the protein comprising said avian lysozyme signal peptide joined to a HCV envelope protein or a part thereof is expressed in a eukaryotic cell. Suitable eukaryotic cells include yeast cells such as Saccharomyces or Hansenula cells. ## Claims ### Claim 1 A recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising a leader peptide defined by the amino acid sequence MRSLLILVLCFLPLAALG (SEQ ID NO:99) joined to an HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV. ### Claim 2 (depends on 1) The recombinant nucleic acid according to claim 1 wherein said leader peptide is joined to a protein of the formula [(A1)a-(PS1)b-(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] wherein: A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is the HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. ### Claim 3 (depends on 2) The recombinant nucleic acid according to claim 2 wherein A has an amino acid sequence chosen from SEQ ID NOs:63-65, 70-72 and 74-82, wherein PS has an amino acid sequence chosen from SEQ ID NOs:66-68 and 83-84 or wherein PS is a dibasic site chosen from Lys-Lys, Arg-Arg, Lys-Arg and Arg-Lys or the monobasic site Lys, and wherein HCVENV is chosen from SEQ ID NOs:85-98 and fragments thereof. ### Claim 4 (depends on 1, 2) The recombinant nucleic acids according to claim 1 or 2 further comprising regulatory elements allowing expression of said protein in a eukaryotic host cell. ### Claim 5 (depends on 1) A vector comprising the recombinant nucleic acid according to claim 1. ### Claim 6 (depends on 5) The vector according to claim 5 which is an expression vector. ### Claim 7 (depends on 5, 6) The vector according to claim 5 or 6 which is an autonomously replicating vector or an integrative vector. ### Claim 8 (depends on 6) The vector according to claim 6 which is chosen from SEQ ID NOs: 20, 21, 32, 35, 36, 39, 40, 49 and 50. ### Claim 9 (depends on 1) An isolated yeast cell comprising the recombinant nucleic acid according to claim 1. ### Claim 10 (depends on 9) An isolated yeast cell according to claim 9, which is expressing the recombinant nucleic acid. ### Claim 11 (depends on 9, 10) An isolated yeast cell according to claim 9 or 10 which is expressing the protein characterized by the structure CL-[(A1)a-(PS1)b-(A2)c]-HCVENV[(A3)d-(PS2)e-(A4)f] wherein: CL is the leader peptide, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV, a, b, c, d, e and f are 0 or 1 and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. ### Claim 12 (depends on 9, 10) An isolated yeast cell according to any of claims 9 to 10 which is translocating the protein CL-[(A1)a-(PS1)b(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] to the endoplasmic reticulum upon removal of the CL peptide wherein said protein and said CL peptide are derived from the protein characterized by the structure CL-[(A1)a-(PS1)b-(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] wherein: CL is the leader peptide, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. ### Claim 13 (depends on 9, 10) An isolated yeast cell according to any of claims 9 to 10 which is processing the processing sites PS1 and/or P82 in said protein translocated to the endoplasmic reticulum. ### Claim 14 (depends on 13) An isolated yeast cell according to claim 13 which is N-glycosylating said protein translocated to the endoplasmic reticulum and processed at said sites PS1 and/or PS2. ### Claim 15 (depends on 9, 10) An isolated yeast cell according to any of claims 9 to 10 which is N-glycosylating said protein translocated to the endoplasmic reticulum. ### Claim 16 (depends on 9, 10) The yeast cell according to any one of claims 9-10 which is a Saccharomyces cell, a Schizosaccharomyces cell, a Kluyveromyces cell, a Yarrowia cell, a Hansenula cell, a Pichia cell, an Aspergillus cell, a Neurospora cell, or a Schwanniomyces cell, or a mutant cell derived from any thereof. ### Claim 17 (depends on 16) The yeast cell according to claim 16 wherein said Saccharomyces cell is chosen from a Saccharomyces cerevisiae cell, a Saccharomyces kluyveri cell, or a Saccharomyces uvarum cell; wherein said Schizosaccharomyces cell is a Schizosaccharomyces pombe cell; wherein said Kluyveromyces cell is a Kluyveromyces lactis cell; wherein said Yarrowia cell is a Yarrowia lipolytica cell; wherein said Hansenula cell is a Hansenula polymorpha cell; wherein said Pichia cell is a Pichia pastoris cell; wherein said Neurospora cell is a Neurospora crassa cell; wherein said Schwanniomyces cell is a Schwanniomyces occidentalis cell. ### Claim 18 (depends on 1, 2, 5, 6, 7, 8) A method for producing a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV in a yeast cell, said method comprising transforming said yeast cell with the recombinant nucleic acid according to any of claims 1 to 2 or with the vector according to any of claims 5 to 8, and expressing said protein comprising the leader peptide joined to a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV in said yeast cell. ### Claim 19 (depends on 18) The method according to claim 18 wherein said yeast cell is translocating the protein CL-[(A1)a-(PS1)b-(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] to the endoplasmic reticulum upon removal of the CL peptide wherein said protein and said CL peptide are derived from the protein characterized by the structure CL-[(A1 )a-(PS1)b-(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] wherein: CL is a leader peptide, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and P82 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV, a, b, a, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. ### Claim 20 (depends on 18) The method according to claim 18 wherein said yeast cell is processing the processing sites PS1 and/or PS2 in said protein translocated to the endoplasmic reticulum. ### Claim 21 (depends on 20) The method according to claim 20 wherein said yeast cell is N-glycosylating said protein translocated to the endoplasmic reticulum and processed at said sites PS1 and/or PS2. ### Claim 22 (depends on 18) The method according to claim 18 further comprising in vitro processing of the processing sites PS1 and/or PS2. ### Claim 23 (depends on 18) The method according to claim 18 wherein said yeast cell is N-glycosylating said protein translocated to the endoplasmic reticulum. ### Claim 24 (depends on 18) The method according to claim 18 wherein said yeast cell is a Saccharomyces cell, a Schizosaccharomyces cell, a Kluyveromyces cell, a Yarrowia cell, a Hansenula cell, a Pichia cell, an Aspergillus cell, a Neurospora cell, or a Schwanniomyces cell, or a mutant cell derived from any thereof. ### Claim 25 (depends on 18) The method according to claim 18 further comprising cultivation of said yeast cells in a suitable medium to obtain expression of said protein. ### Claim 26 (depends on 25) The method according to claim 25 further comprising isolation of the expressed protein from a culture of said yeast cells, or from said yeast cells. ### Claim 27 (depends on 26) The method according to claims 26 wherein said isolation step involves lysis of said yeast cells in the presence of a chaotropic agent. ### Claim 28 (depends on 26) The method according to claim 26 wherein the cysteine thiol-groups in the isolated proteins are chemically modified and wherein said chemical modification is reversible or irreversible. ### Claim 29 (depends on 25) The method according to claim 25 involving heparin affinity chromatography. ### Claim 30 (depends on 1, 2, 5, 6, 7, 8) A method for producing a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV in a yeast cell, said method comprising transforming said yeast cell with the recombinant nucleic acid according to any of claims 1 to 2 or with the vector according to any of claims 5 to 8, and expressing said protein characterized by the structure CL-[(A1)a-(PS1)b(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] in said host cell wherein: CL is a leader peptide, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. ### Claim 31 (depends on 7) A method for producing a HCV envelope protein or the sequence of SEQ ID NO: 2 or a corresponding sequence from another HCV in a yeast cell, said method comprising transforming said yeast cell with the vector according to claim 7, and expressing said protein comprising the leader peptide joined to a HCV envelope protein or to the sequence of SEQ ID NO: 2 or a corresponding sequence from another HCV in said yeast cell. ### Claim 32 (depends on 7) A method for producing a HCV envelope protein or the sequence of SEQ ID NO: 2 or a corresponding sequence from another HCV in a yeast cell, said method comprising transforming said yeast cell with the vector according to claim 7, and expressing said protein characterized by the structure CL-[(A1)a-(PS1)b-(A2)c]-HCVENV-[(A3)d-(PS2)e-(A4)f] in said yeast cell wherein: CL is a leader peptide, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or the sequence of SEQ ID NO: 2 or a corresponding sequence from another HCV, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.