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

• US 9,012,199 B2
• Filed: 10/23/2013
• Issued: 04/21/2015
• Est. Priority Date: 05/22/2003
• Status: Active Grant

• Current Assignee: iBio, Inc.
• Original Assignee: iBio, Inc.
• Inventors: Yusibov, Vidadi, Mett, Vadim, Musiychuk, Konstantin
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US14/060,793
• Publication Number:
  US 20140141508A1
• Time in Patent Office: 545 Days
• Field of Search: None
• US Class Current: 435/252.3
• 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 nucleic acid that encodes a carrier protein comprising a recombinant carrier molecule, wherein the recombinant carrier molecule has at least 90% amino acid identity to an amino acid sequence of a modified lichenase B (licB) polypeptide that is based on a rearranged amino acid sequence of the Clostridium thermocellum wild-type licB (wild-type licB) amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO:

18, wherein the modified licB polypeptide comprises original N-terminal and C-terminal regions that correspond in sequence to N- and C-terminal polypeptides obtained by splitting the amino acid sequence encoded by SEQ ID NO;

18 within the loop region defined by amino acid residues 82-94 of the amino acid sequence encoded by SEQ ID NO;

18, wherein the modified licB polypeptide comprises the original C-terminal region fused upstream of the original N-terminal region, and wherein the recombinant carrier molecule optionally has a heterologous polypeptide comprising a therapeutic polypeptide or a disease-associated epitope fused thereto.

2. The nucleic acid of claim 1, wherein the modified lichenase B polypeptide 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.

3. The nucleic acid of claim 2, wherein the nucleic acid comprises a nucleic acid that encodes the heterologous polypeptide inserted into or fused in frame with a nucleic acid whose sequence comprises SEQ ID NO:

1 or 5.

4. An expression vector comprising the nucleic acid of claim 2.

5. An expression vector comprising the nucleic acid of claim 2, wherein the vector is a plant virus vector.

6. A host cell comprising the expression vector of claim 4.

7. The host cell of claim 6, wherein the host cell is transformed with the expression vector.

8. The host cell of claim 6, wherein the host cell is selected from the group consisting of plant cells, bacterial cells, insect cells, yeast cells, and mammalian cells.

9. The nucleic acid of claim 1, wherein the heterologous polypeptide is fused to the N-terminus or C-terminus of the recombinant carrier molecule.

10. The nucleic acid of claim 1, wherein the heterologous polypeptide is fused at an internal location of the recombinant carrier molecule.

11. The nucleic acid of claim 1, wherein the recombinant carrier molecule is fused to two or more heterologous polypeptides.

12. An expression vector comprising the nucleic acid of claim 1.

13. The expression vector of claim 12, wherein the vector is a plant virus vector.

14. A host cell comprising the expression vector of claim 12.

15. The host cell of claim 14, wherein the host cell is selected from the group consisting of plant cells, bacterial cells, insect cells, yeast cells, and mammalian cells.

16. The nucleic acid of claim 1, wherein the heterologous polypeptide sequence comprises a vaccine antigen.

17. An expression vector comprising the nucleic acid of claim 16.

18. The expression vector of claim 17, wherein the vector is a plant virus vector.

19. A host cell comprising the expression vector of claim 17.

20. The host cell of claim 19, wherein the host cell is selected from the group consisting of plant cells, bacterial cells, insect cells, yeast cells, and mammalian cells.