Fusion proteins containing luciferase and a polypeptide of interest

Patent metadata

Field	Value
Publication number	US-10829770-B2
Application number	US-201916571616-A
Country	US
Kind code	B2
Filing date	Sep 16, 2019
Priority date	Sep 8, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

Polynucleotides encoding fusion proteins contain a secretable luciferase fused to a modified polypeptide of interest are disclosed. The polypeptide of interest has been modified to remove a native N-terminal secretion sequence and has been replaced by the secretable luciferase. One example of a modified polypeptide of interest is interferon. The polynucleotides and fusion proteins have biotherapeutic, diagnostic, and quality control applications in biotechnological, medical, and veterinary fields. Methods for producing the secretable fusion protein are also disclosed.

First claim

What is claimed is:

1. A polynucleotide comprising a single open reading frame (ORF) that encodes a fusion protein comprising a luciferase, an interferon, and an Aphthovirus 2A.

2. The polynucleotide of claim 1 , wherein the fusion protein comprises, in order from the N-terminal, the luciferase, the Aphthovirus 2A and the interferon.

3. The polynucleotide of claim 1 , wherein the fusion protein comprises, in order from the N-terminal, the interferon, the Aphthovirus 2A and the luciferase.

4. The polynucleotide of claim 3 , wherein the amino acid sequence of the luciferase is modified to remove its N-terminal methionine initiation site.

5. The polynucleotide of claim 1 , wherein the Aphthovirus 2A comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16 or SEQ ID NO: 18.

6. The polynucleotide of claim 1 , comprising the nucleotide sequence of SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 108 or SEQ ID NO: 109.

7. The polynucleotide of claim 1 , wherein the Aphthovirus 2A is FMDV Δ1D2A.

8. The polynucleotide of claim 1 , wherein the Aphthovirus 2A comprises at least one selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22.

9. A polynucleotide comprising a single open reading frame (ORF) that encodes a fusion protein comprising, in order from the N-terminal, a secretable luciferase fused to a modified polypeptide of interest, wherein:
the modified polypeptide of interest has been modified to remove a native N-terminal secretion peptide sequence;
the removed N-terminal secretion peptide sequence has been replaced by the secretable luciferase; and
the polynucleotide encoding the secretable luciferase is directly adjoined at the 3′ end to the 5′ end of the polynucleotide encoding the modified polypeptide of interest.

10. The polynucleotide of claim 9 , further comprising at least one promoter or other transcription regulatory element.

11. The polynucleotide of claim 9 , wherein the polypeptide of interest is an interferon.

12. The polynucleotide of claim 9 , encoding for the polypeptide of at least one selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 111, and SEQ ID NO: 113.

13. A fusion protein comprising, in order from the N-terminal, a secretable luciferase and a modified polypeptide of interest, wherein:
the modified polypeptide of interest has been modified to remove a native N-terminal secretion peptide sequence;
the removed N-terminal secretion peptide domain has been replaced by the secretable luciferase; and
the secretable luciferase is directly adjoined at the C-terminus to the N-terminus of the modified polypeptide of interest.

14. The fusion protein of claim 13 , wherein the polypeptide of interest is an interferon.

15. The fusion protein of claim 13 , comprising at least one selected from the group consisting of: SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 111, and SEQ ID NO: 113.

16. A method for producing a secretable fusion protein, comprising:
providing a host cell expressing a polynucleotide comprising a single open reading frame encoding the fusion protein;
culturing the host cell in a suitable medium, wherein the secretable fusion protein is expressed and secreted by the host cell into the medium; and
recovering the secreted fusion protein from the medium,
wherein:
the fusion protein comprises, in order from the N-terminal, a secretable luciferase and a modified polypeptide of interest;
the modified polypeptide of interest has been modified to remove a native N-terminal secretion peptide domain;
the native N-terminal secretion peptide domain has been replaced by the secretable luciferase; and
the luciferase is directly adjoined at the C-terminus to the N-terminus of the modified polypeptide of interest.

17. The method of claim 16 , wherein the host cell is a eukaryotic cell.

18. The method of claim 16 , wherein the polypeptide of interest is an interferon.

19. The method of claim 16 , wherein the polynucleotide comprises at least one promoter or other transcription regulatory element.

Assignees

• The Government Of The Us Secretary Of Homeland Security

Inventors

• Puckette Michael
• Rasmussen Max V

Classifications

• C12Q1/66

  involving luciferase · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C07K14/56

  IFN-alpha · CPC title

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• A61K38/00

  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) · CPC title