Constructs and methods for expression of recombinant HCV envelope proteins
View Patent ↗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.
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.
2. 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.
3. 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.
4. The recombinant nucleic acids according to claim 1 or 2 further comprising regulatory elements allowing expression of said protein in a eukaryotic host cell.
5. A vector comprising the recombinant nucleic acid according to claim 1 .
6. The vector according to claim 5 which is an expression vector.
7. The vector according to claim 5 or 6 which is an autonomously replicating vector or an integrative vector.
8. The vector according to claim 6 which is chosen from SEQ ID NOs: 20, 21, 32, 35, 36, 39, 40, 49 and 50.
9. An isolated yeast cell comprising the recombinant nucleic acid according to claim 1 .
10. An isolated yeast cell according to claim 9 , which is expressing the recombinant nucleic acid.
11. 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.
12. 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.
13. 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.
14. 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.
15. An isolated yeast cell according to any of claims 9 to 10 which is N-glycosylating said protein translocated to the endoplasmic reticulum.
16. 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.
17. 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.
18. 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.
19. 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.
20. 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.
21. 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.
22. The method according to claim 18 further comprising in vitro processing of the processing sites PS1 and/or PS2.
23. The method according to claim 18 wherein said yeast cell is N-glycosylating said protein translocated to the endoplasmic reticulum.
24. 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.
25. The method according to claim 18 further comprising cultivation of said yeast cells in a suitable medium to obtain expression of said protein.
26. 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.
27. The method according to claims 26 wherein said isolation step involves lysis of said yeast cells in the presence of a chaotropic agent.
28. 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.
29. The method according to claim 25 involving heparin affinity chromatography.
30. 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.
31. 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.
32. 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.