Recombinant carrier molecule for expression, delivery and purification of target polypeptides

• US 8,173,408 B2
• Filed: 11/24/2009
• Issued: 05/08/2012
• Est. Priority Date: 05/22/2003
• Status: Active Grant

• Current Assignee: iBio, Inc.
• Original Assignee: Fraunhofer USA Inc. (Fraunhofer-Institut für Nachrichtentechnik HHI)
• Inventors: Yusibov, Vidadi, Mett, Vadim, Musiychuk, Konstantin
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US12/625,129
• Publication Number:
  US 20100227373A1
• Time in Patent Office: 896 Days
• Field of Search: None
• US Class Current: 435/200
• CPC Class Codes: A61K 2039/6031 Proteins
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 16/1027 Paramyxoviridae, e.g. respiratory syncytial virus
• CPC Class Codes: C07K 16/1278 from Bacillus (G)
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/2448 Licheninase (3.2.1.73)
• CPC Class Codes: C12Y 302/01073 Licheninase (3.2.1.73)

Abstract

Recombinant carrier molecules having amino acid sequences from thermostable enzymes and methods of use for expression, recovery and delivery of foreign sequences (peptides and polypeptides) produced in different systems (bacteria, yeast, DNA, cell cultures such as mammalian, plant, insect cell cultures, protoplast and whole plants in vitro or in vivo are provided. The recombinant carrier molecule using sequences from lichenase B(Lic B) were also made and used as part of carrier protein to express, recover and deliver a variety of target polypeptides of interest.

Claims

1. A recombinant polypeptide comprising a modified lichenase B polypeptide that has an amino acid sequence with at least 90% sequence identity to amino acids 14-231 of SEQ ID NO:

3, or an amino acid sequence with at least 90% sequence identity to amino acids 8-226 of SEQ ID NO;

6, wherein the modified lichenase B polypeptide is optionally fused to a heterologous polypeptide sequence that is not found in the modified lichenase B polypeptide.

2. The recombinant polypeptide of claim 1, wherein the modified lichenase B polypeptide is encoded by SEQ ID NO:

1 or 5.

3. The recombinant polypeptide of claim 1, wherein the modified lichenase B polypeptide comprises the sequence set forth in SEQ ID NO:

3 or SEQ ID NO;

6.

4. The recombinant polypeptide of claim 1, wherein the heterologous polypeptide sequence is fused to the modified lichenase B polypeptide so as to create a fusion protein.

5. The recombinant polypeptide of claim 1, wherein the modified lichenase B polypeptide has an amino acid sequence with at least 95% sequence identity to amino acids 14-231 of SEQ ID NO:

3, or at least 95% sequence identity to amino acids 8-226 of SEQ ID NO;

6.

6. The recombinant polypeptide of claim 1, wherein the modified lichenase B polypeptide has the sequence of amino acids 14-231 of SEQ ID NO:

3, or the amino acid sequence of amino acids 8-226 of SEQ ID NO;

6.

7. The recombinant polypeptide of claim 4, wherein the modified lichenase B polypeptide is fused to two or more heterologous epitope-containing polypeptide segments so as to create a fusion protein.

8. The recombinant polypeptide of claim 4, wherein the heterologous polypeptide is fused to the N-terminus or C-terminus of the modified lichenase B polypeptide, or is fused internally into the modified lichenase B polypeptide.

9. A method comprising administering to an animal a composition comprising the recombinant polypeptide of claim 1, wherein the recombinant polypeptide comprises:

(a) a modified lichenase B polypeptide that has an amino acid sequence with at least 90% sequence identity to amino acids 14-231 of SEQ ID NO;

3, or an amino acid sequence with at least 90% sequence identity to amino acids 8-226 of SEQ ID NO;

6, fused to

(b) a heterologous polypeptide sequence that is not found in the lichenase B polypeptide, wherein the heterologous polypeptide comprises at least one disease-associated antigen, wherein the composition further comprises a pharmaceutically acceptable carrier medium or adjuvant, and wherein the administering stimulates an immune response to the at least one disease-associated antigen in the animal.

10. The method of claim 9, wherein the recombinant polypeptide has lichenase activity.

11. The method of claim 9, wherein the modified lichenase B polypeptide has an amino acid sequence with at least 95% sequence identity to amino acids 14-231 of SEQ ID NO:

3, or an amino acid sequence with at least 95% sequence identity to amino acids 8-226 of SEQ ID NO;

6.

12. The method of claim 9, wherein the heterologous polypeptide is fused to the N-terminus or C-terminus of the modified lichenase B polypeptide, or is fused internally into the modified lichenase B polypeptide.

13. The recombinant polypeptide of claim 1, wherein the heterologous polypeptide sequence comprises a growth factor, cytokine, ligand, receptor, inhibitor, antigen, epitope, T cell epitope, B cell epitope, antibody, hydrolase, green fluorescent protein, interferon, interleukin, pathogen-associated protein, or toxin.

14. The recombinant polypeptide of claim 1, wherein the heterologous polypeptide sequence comprises a vaccine antigen.

15. The recombinant polypeptide of claim 1, further comprising a tag usable for separation or purification, or both.

16. The recombinant polypeptide of claim 15, wherein the tag is His 6.

17. The recombinant polypeptide of claim 16, further comprising an enzymatic cleavage site.

18. The recombinant polypeptide of claim 17, wherein the cleavage site is located between the heterologous polypeptide and the modified lichenase B polypeptide.

19. The recombinant polypeptide of claim 1, further comprising a tag and a cleavage site, wherein the cleavage site is located adjacent to the tag.

20. The recombinant polypeptide of claim 1, further comprising a linker.