Canadian Patents Database

Claims page

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Patent Document Number: 2495145

(54) English Title: STAPHYLOCOCCAL NUCLEASE FUSION PROTEINS FOR THE PRODUCTION OF RECOMBINANT PEPTIDES

(54) French Title: NOUVELLES PROTEINES HYBRIDES ISSUES DE LA NUCLEASE STAPHYLOCOCCIQUE ET DESTINEES A LA PRODUCTION DE PEPTIDES RECOMBINANTS

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Claims:

Note: Claims are shown in the official language in which they were submitted.


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WHAT IS CLAIMED IS:

1. A fusion carrier protein for expressing a target peptide, said
fusion carrier protein being derived from Staphylococcus
nuclease, or a mutant thereof, and consisting of between 80
and 120 amino acid in length.
2. A fusion carrier protein having an amino acid sequence as set
forth in Formula I:
T1-A1-X1-A2-X2-A3-X3-A3-X4-A5-A6-A7-X5-A8-A9-T2 (I)
Wherein
T1 is absent, a His-tag or at least one peptidic cleavage site,
A1 is Ala-Thr-Ser-Thr-Lys-Lys-Leu-His-Lys-Glu-Pro-Ala-Thr-
Leu-Ile-Lys-Ala-Ile-Asp-Gly-Asp-Thr-Val-Lys-Leu (SEQ ID
NO:1),
X1, X2, X3, X4, and X5, each independently is any one amino
acid or a His-tag,
A2 is Tyr-Lys-Gly-Gln-Pro (SEQ ID NO:2),
A3 is Leu-Leu-Leu-Val-Asp-Thr-Pro-Glu-Thr-Lys-His-Pro-
Lys-Lys-Gly-Val-Glu-Lys-Tyr-Gly-Pro-Glu-Ala-Ser-Ala-
Phe-Thr-Lys-Lys (SEQ ID NO:3),
A4 is Val-Glu-Asn-Ala-Lys-Lys-Ile-Glu-Val-Glu-Phe-Asp-Lys-
Gly-Gln-Arg-Thr-Asp-Lys-Tyr-Gly-Arg-Gly-Leu-Ala-Tyr-Ile-
Tyr-Ala-Asp-Gly-Lys (SEQ ID NO:4),
A5 is Val-Asn-Glu-Ala-Leu (SEQ ID NO:5),
A6 is absent or at lest one of Asp-Pro, Phe-Asn-Pro-Arg-Gly-
Ser (SEQ ID NO:6) and His-tag,


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A7 is absent or Val-Arg-Gln-Gly-Leu-Ala-Lys-Val-Ala-Tyr-Val-
Tyr-Lys-Pro (SEQ ID NO:7),
A8 is absent or at least one of Asp-Pro and Phe-Asn-Pro-
Arg-Gly-Ser (SEQ ID NO:6),
A9 is absent or Asn-Asn-Thr-His-Glu-Gln-Leu-Leu-Arg-Lys-
Ser-Glu-Ala-Gln-Ala-Lys-Lys-Glu-Lys-Leu-Asn-Ile-Trp-
Ser-Glu-Asp-Asn-Ala-Asp-Ser-Gly-Gln (SEQ ID NO:8),
and
T2 is absent, a His-tag or at least one peptidic cleavage site.
3. The fusion carrier protein of claim 1, wherein the peptidic
cleavage site is selected from the group consisting of Met, Asp-
Pro, Gly-Pro, Asp-Gly, Phe-Asn-Pro-Arg (SEQ ID NO:9), Leu-
Val-Pro-Arg (SEQ ID NO:10), Phe-Asn-Pro-Arg-Gly-Ser (SEQ
ID NO:6), and Asp-Asp-Asp-Asp-Lys (SEQ ID NO:12).
4. The fusion carrier protein of claim 1, wherein the His-tag is
composed of three to eight histidine residues.
5. A fusion carrier protein comprising a sequence as set forth in
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20
or SEQ ID NO:24.
6. A fusion protein comprising the fusion carrier protein as defined
in claim 1, 2, 3, 4 or 5, linked to at least one target peptide.
7. The fusion protein of claim 6, wherein the target peptide is
linked to the C-terminus of the fusion carrier protein.
8. The fusion protein of claim 6, wherein the target peptide is
linked to the N-terminus of the fusion carrier protein.
9. The fusion protein of claim 6, 7 or 8, wherein the target peptide
has a sequence between 2 and 100 amino acids in length.


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10. The fusion protein of claim 6, 7, 8 or 9, wherein the target
peptide is selected from the group of peptide consisting of
eCla4, eSte20, hirudin, mCla4, mSte20, cCla4, cSte20, FpA,
FD22, propeptide of human Cathepsin B, PTH and EphrinB, or
fragments thereof.
11. The fusion protein of claim 6, 7, 8, 9, 10, further comprising a
peptidic cleavage site between the fusion carrier protein and the
target peptide.
12. A nucleic acid sequence encoding the fusion protein of claim 6,
7, 8, 9, 10 or 11.
13. An expression vector comprising the nucleic acid sequence of
claim 12, operably linked to a promoter for expression of said
nucleic acid sequence coding for the fusion protein.
14. The expression vector of claim 13, wherein the promoter is pL
promoter, .lambda. promoter, trc promoter or T7 promoter.
15. A host cell transformed with the expression vector of claim 13 or
14.
16. The host cell of claim 15, wherein said host cell is E. coli DH5.alpha.,
BL21, JM101 or JM105 or NM522 or N99Cl+.
17. The host cell of claim 15, wherein said host cell is from E. coli or
B. subtilis.
18. The host cell of claim 15, wherein said host cell is a yeast.
19. A method for producing a fusion protein comprising the step of
culturing the host cell as defined in claim 15, 16, 17 or 18 under
suitable conditions for expression of the expression vector,
thereby producing a fusion protein.
20. The method of claim 19, wherein the suitable conditions
comprise an inducer for inducing the host cell to express the
espression vector.


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21. The method of claim 20, wherein the inducer is IPTG, nalidixic
acid or temperature.
22. The method of claim 19, 20 or 21, further comprising a step of
purification of the fusion protein produced.
23. The method of claim 22, wherein the step of purification
comprises at least one of alcohol precipitation, ion-exchange,
and affinity purification using Ni-agarose resin.
24. The method of claim 19, 20, 21, 22, or 23, wherein the fusion
protein is further subjected to a proteolytic digestion to release
the target peptide from the fusion protein.
25. The method of claim 24, wherein the proteolytic digestion is
achieved by CNBr, formic acid or HCl.
26. The method of claim 24, wherein the proteolytic digestion is
achieved by thrombin, or a protease.
27. The method of claim 26, wherein the protease is an
enterokinase.
28. The method of claim 24, 25, 26 or 27, wherein the target
peptide released is further purified by HPLC.
29. Use of a fusion carrier protein as defined in claim 1, 2, 3, 4 or 5
for expressing a target peptide.
30. Use of a nucleic acid sequence as defined in claim 12 for
expressing a target peptide.
31. Use of an expression vector as defined in claim 13 or 14 for
expressing a target peptide.
32. Use of a host cell as defined in claim 15, 16, 17 or 18 for
expressing a target peptide.