Synthesis of human procollagens and collagens in recombinant DNA systems

• US 20020142391A1
• Filed: 12/19/2001
• Published: 10/03/2002
• Est. Priority Date: 06/12/1991
• Status: Abandoned Application

• Current Assignee: Kari I. Kivirikko, Taina Pihlajaniemi
• Original Assignee: Kari I. Kivirikko, Taina Pihlajaniemi
• Inventors: Pihlajaniemi, Taina, Kivirikko, Kari I.
• Application Number:
  US10/040,204
• Publication Number:
  US 20020142391A1
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0325 Animal model for autoimmune diseases
• CPC Class Codes: A61K 38/00 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 Class Codes: A61K 49/0008 Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12Y 114/11002 Procollagen-proline dioxygenase (1.14.11.2), i.e. proline-hydroxylase

Abstract

Methods of making collagen with hosts, and vectors that express collagen, and collagen post-translation enzymes are disclosed. Collagen post-translation enzymes include prolyl-4-hydroxylase, lysyl hydroxylase, lysyl oxidase, C-proteinase, and N-proteinase, and these enzymes increase the yield of properly folded, recombinant collagen in non-mammalian hosts. The collagens produced by these methods, hosts, and vectors include both homotrimer and heterotrimer collagen made from single or multiple collagen genes, respectively.

Claims

1. A method for producing a collagen polypeptide, wherein said collagen is selected from the group comprising collagen types IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX, comprising:

a. culturing a host cell, wherein said host cell has been infected, transfected or transformed with (i) a first expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a collagen subunit; and

(ii) a second expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes at least one collagen post-translational enzyme or subunit thereof; and

b. purifying said collagen polypeptide.

2. The method of claim 1 wherein the host cell is selected from the group consisting of a yeast cell, a plant cell, an insect cell and a mammalian cell.

3. The method of claim 1 wherein the host cell is further infected, transfected or transformed with a third expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a second collagen subunit.

4. The method of claim 3 wherein the host cell is further infected, transfected or transformed with a fourth expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a third collagen subunit.

5. The method of claim 1 wherein said collagen post-translational enzyme is selected from the group consisting of prolyl-4-hydroxylase, lysyl oxidase, lysyl hydroxylase, C-proteinase, and N-proteinase.

6. The method of claim 1 wherein the collagen post-translational enzyme subunit is selected from the group consisting of an alpha subunit of prolyl-4-hydroxylase and a beta subunit of prolyl-4-hydroxylase.

7. A method for producing a procollagen polypeptide, wherein said procollagen is selected from the group comprising collagen types IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX, comprising:

a. culturing a host cell, wherein said host cell has been infected, transfected or transformed with;

(i) a first expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a collagen subunit; and

(ii) a second expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes at least one collagen post-translational enzyme or subunit thereof; and

b. purifying said procollagen polypeptide.

8. The method of claim 7 wherein the host cell is selected from the group consisting of a yeast cell, a plant cell, an insect cell and a mammalian cell.

9. The method of claim 7 wherein the host cell is further infected, transfected or transformed with a third expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a second collagen subunit.

10. The method of claim 9 wherein the host cell is further infected, transfected or transformed with a fourth expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a third collagen subunit.

11. The method of claim 7 wherein said collagen post-translational enzyme is selected from the group consisting of prolyl-4-hydroxylase, lysyl oxidase and lysyl hydroxylase.

12. The method of claim 7 wherein the collagen post-translational enzyme subunit is selected from the group consisting of an alpha subunit of prolyl-4-hydroxylase and a beta subunit of prolyl-4-hydroxylase.

14. The collagen polypeptide of claim 13 wherein the host cell is selected from the group consisting of a yeast cell, a plant cell, an insect cell and a mammalian cell.

15. The collagen polypeptide of claim 13 wherein the host cell is further infected, transfected or transformed with a third expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a second collagen subunit.

16. The collagen polypeptide of claim 15 wherein the host cell is further infected, transfected or transformed with a fourth expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a third collagen subunit.

17. The collagen polypeptide of claim 13 wherein said collagen post-translational enzyme is selected from the group consisting of prolyl-4-hydroxylase, lysyl oxidase, lysyl hydroxylase, C-proteinase, and N-proteinase.

18. The collagen polypeptide of claim 13 wherein the collagen post-translational enzyme subunit is selected from the group consisting of an alpha subunit of prolyl-4-hydroxylase and a beta subunit of prolyl-4-hydroxylase.

19. The collagen polypeptide of claim 13 wherein said polypeptide is not glycosolated.

20. The collagen polypeptide of claim 13 wherein said polypeptide is partially deglycosolated.

13. A collagen polypeptide, wherein said collagen is selected from the group comprising collagen types IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX, manufactured according to a method comprising:

a. culturing a host cell, wherein said host cell has been infected, transfected or transformed with;

(i) a first expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a collagen subunit; and

(ii) a second expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes at least one collagen post-translational enzyme or subunit thereof; and

b. purifying said collagen polypeptide.

21. A host cell which has been infected, transfected or transformed with:

(i) a first expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes a collagen subunit; and

(ii) a second expression vector comprising a polynucleotide molecule having a nucleic acid sequence which encodes at least one collagen post-translational enzyme or subunit thereof.

22. The host cell of claim 21 wherein said host cell is further infected, transfected or transformed with a third expression vector comprising a second collagen subunit.

23. The host cell of claim 22 wherein said host cell is further infected, transfected or transformed with a fourth expression vector comprising a third collagen subunit.