Signal sequences to improve protein expressions and secretion of recombinant enzymes and other proteins

Patent metadata

Field	Value
Publication number	US-9279007-B2
Application number	US-201113988946-A
Country	US
Kind code	B2
Filing date	Nov 22, 2011
Priority date	Nov 22, 2010
Publication date	Mar 8, 2016
Grant date	Mar 8, 2016

Abstract

Polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) are disclosed. Also disclosed are fusion proteins comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide. Also disclosed are protein expression vectors comprising a promoter operably linked to a first DNA sequence encoding a signal sequence comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a second DNA sequence encoding a heterologous polypeptide fused in frame to the first DNA sequence. Further disclosed are methods of producing a polypeptide comprising expressing a fusion protein comprising a polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide and recovering the heterologous polypeptide.

First claim

What is claimed is:

1. A signal polypeptide comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip), wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) has the amino acid sequence of SEQ ID NO: 19 with an insertion of seven to ten amino acid residues after position 13 of SEQ ID NO: 19, and wherein the last four inserted amino acid residues are leucine residues, and wherein the signal polypeptide is effective for a protein translocation when fused to the protein.

2. The signal polypeptide of claim 1 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) has the amino acid sequence of SEQ ID NO: 20.

3. The signal polypeptide of claim 1 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) has the amino acid sequence of SEQ ID NO: 21.

4. The signal polypeptide of claim 1 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) has the amino acid sequence of SEQ ID NO: 22.

5. The signal polypeptide of claim 1 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) has the amino acid sequence of SEQ ID NO: 23.

6. A fusion protein comprising the signal polypeptide of claim 1 operably linked to a heterologous polypeptide.

7. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has amino acid sequence of SEQ ID NO: 20.

8. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 21.

9. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 22.

10. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 23.

11. The fusion protein of claim 6 wherein the heterologous polypeptide comprises one or more enzymes, one or more biological response modifiers, one or more toxins, one or more antibodies, one or more fragments of the heterologous polypeptide, or any combination thereof.

12. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 20 and the heterologous polypeptide comprises one or more of the following: acid β-glucocerebrosidase, acid α-galactosidase, acid α-glucosidase, proinsulin, insulin-like growth hormone-2 (IGF-2), interferon, therapeutic antibody and an insulin-like growth hormone-1 (IGF-1).

13. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 21 and the heterologous polypeptide comprises one or more of the following: acid β-glucocerebrosidase, acid α-galactosidase, acid α-glucosidase, proinsulin, insulin-like growth hormone-2 (IGF-2), interferon, therapeutic antibody or an insulin-like growth hormone-1 (IGF-1).

14. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 22 and the heterologous polypeptide comprises one or more of the following: acid β-glucocerebrosidase, acid α-galactosidase, acid α-glucosidase, proinsulin, insulin-like growth hormone-2 (IGF-2), interferon, therapeutic antibody or an insulin-like growth hormone-1 (IGF-1).

15. The fusion protein of claim 6 wherein the modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence has the amino acid sequence of SEQ ID NO: 23 and the heterologous polypeptide comprises one or more of the following: acid β-glucocerebrosidase, acid α-galactosidase, acid α-glucosidase, proinsulin, insulin-like growth hormone-2 (IGF-2), interferon, therapeutic antibody or an insulin-like growth hormone-1 (IGF-1).

16. A method of producing a polypeptide, comprising:
(a) expressing a fusion protein comprising the signal polypeptide of claim 1 operably linked to a heterologous polypeptide; and
(b) recovering said heterologous polypeptide.

17. A method of increasing protein expression, comprising:
(a) expressing a fusion protein comprising the signal polypeptide of claim 1 and a heterologous protein, and
(b) recovering the heterologous protein.

18. A method of increasing protein secretion, comprising:
(a) expressing a fusion protein comprising the signal polypeptide of claim 1 and a heterologous protein, and
(b) recovering the heterologous protein.

Assignees

• Do Hung
• Amicus Therapeutics Inc

Inventors

• Do Hung

Classifications

• C07K14/47 Primary

  from mammals · CPC title

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C12N9/0002

  Antibodies with enzymatic activity, e.g. abzymes · CPC title

• C12N9/2434

  acting on beta-1,4-glucosidic bonds · CPC title

• C12N15/85

  for animal cells · CPC title