Polyfunctional base sequence and artificial gene containing the same

• US 20030121065A1
• Filed: 12/13/2002
• Published: 06/26/2003
• Est. Priority Date: 06/16/2000
• Status: Abandoned Application

• Current Assignee: Japan Science And Technology Agency
• Original Assignee: Japan Science And Technology Agency
• Inventors: Shiba, Kiyotaka
• Application Number:
  US10/311,111
• Publication Number:
  US 20030121065A1
• Time in Patent Office: Days
• US Class Current: 800/8
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

Abstract

The present invention provides: a microgene comprising a multifunctional base sequence having two or more functions in different reading frames of the base sequence which is required for creating industrially useful artificial proteins that do not exist in nature; an artificial gene obtained by polymerizing the microgenes; and an artificial protein which is a translation product of the artificial gene.

A microgene is produced by selecting a base sequence by the computational science approach from among all combinations of base sequences encoding an amino acid sequence having a given function where the selected base sequence has a biological function same as or different from the given function in a reading frame different from that of the amino acid sequence having the given function, and is devoid of termination codons in all three reading frames; the microgene thus obtained is then polymerized in such a manner as plural reading frames emerge to produce an artificial gene; and the artificial protein as a translation product of the artificial gene is obtained.

Claims

1. A multifunctional base sequence wherein the base sequence has two or more functions in different reading frames of the base sequence.

2. The multifunctional base sequence according to claim 1, wherein the base sequence is a double-stranded base sequence.

3. The multifunctional base sequence according to claim 1 or 2, wherein the base sequence is DNA.

4. The multifunctional base sequence according to claim 1 or 2, wherein the base sequence is RNA.

5. The multifunctional base sequence according to any of claims 1-4, wherein the base sequence is a linear base sequence.

6. The multifunctional base sequence according to any of claims 1-5, wherein all three reading frames emerging from the one-by-one frameshifts of reading frames in the base sequence are devoid of termination codons.

7. The multifunctional base sequence according to any of claims 2-6, wherein all six reading frames of the base sequence are devoid of termination codons.

8. The multifunctional base sequence according to claim 6 or 7, wherein termination codons do not emerge at the junction points arising from the polymerization of the multifunctional base sequence.

9. The multifunctional base sequence according to any of claims 1-8, wherein the two or more functions are two or more biological functions.

10. The multifunctional base sequence according to claim 9, wherein the biological functions are:

function of easily forming secondary structures;

antigen function to induce neutralizing antibodies;

function to activate immunity;

function to promote or suppress cell proliferation;

function to specifically recognize cancer cells;

protein transduction function;

cell-death-inducing function;

function to present residues that determine antigens;

metal-binding function;

coenzyme-binding function;

function to activate catalysts;

function to activate fluorescence signal;

function to bind to a specific receptor and to activate the receptor;

function to bind to a specific factor involved in signal transduction and to modulate the action of the factor;

function to specifically recognize biopolymers;

cell adhesion function;

function to localize proteins to the cell exterior;

function to target at a specific intracellular organelle;

function to be embedded in the cell membrane;

function to form amyloid fibers;

function to form fibrous proteins;

function to form a protein gel;

function to form a protein film;

function to form a single molecular membrane;

self-aggregation function;

function to form particles;

or function to assist the formation of higher-order structure of other proteins.

11. The multifunctional base sequence according to any of claims 1-10, wherein the base sequence comprises 15-500 bases or base pairs.

12. The multifunctional base sequence according to any of claims 1-11, wherein the multifunctional base sequence is modified for polymerization.

13. The multifunctional base sequence according to any of claims 1-12, wherein a natural base sequence is linked thereto.

30. An artificial gene to which one or more multifunctional base sequences according to any of claims 1-13 or to claim 29 are bound in such a manner as plural reading frames emerge.

31. The artificial gene according to claim 30 which comprises 30-100000 bases or base pairs.

32. A method of producing an artificial gene wherein one or more multifunctional base sequences according to any of claims 1-13 or to claim 29 are combinatorially polymerized in such a manner as plural reading frames emerge.

33. The method of producing an artificial gene according to claim 32 which comprises:

adding a specific DNA sequence [A] at the one end of a double-stranded multifunctional base sequence;

adding a specific DNA sequence [B] at the another end of the base sequence;

preparing DNA sequences [a] and [b] which are at least partially complementary to DNA sequences [A] and [B] respectively; and

performing a ligase reaction for the double-stranded multifunctional base sequence by using a single-stranded DNA sequence to which the DNA sequences [a] and [b] are linked.

34. The method of producing an artificial gene according to claim 32, wherein polymerase is made to act on two base sequences, which are comprised of the whole or a part of a multifunctional base sequence and which are at least partially complementary to each other, for the polymerase chain reaction to occur.

35. An artificial gene expression vector wherein the artificial gene according to claim 30 or 31 is incorporated in an expression vector.

36. The artificial gene expression vector according to claim 35, wherein the artificial gene is bound to a natural gene.

37. A cell containing an artificial gene expression vector, wherein the artificial gene expression vector according to claim 35 or 36 is introduced into a host cell.

38. The cell containing an artificial gene expression vector according to claim 37, wherein the host cell is E. coli.

39. An artificial protein or its derivative obtained as a translation product of the artificial gene according to claim 30 or 31.

40. The artificial protein or its derivative according to claim 39, wherein the translation takes place in a cell-free expression system.

41. The artificial protein or its derivative according to claim 39, wherein the translation takes place in a cell expression system.

42. The artificial protein or its derivative according to claim 41, wherein the cell is the cell containing an artificial gene expression vector according to claim 37 or 38.

43. The artificial protein or its derivative according to any of claims 39-42, wherein a derivative of the artificial protein is:

artificial glycoprotein;

artificial phospholipid protein;

artificial polyethylene glycol-modified protein;

artificial porphyrin-binding protein;

or artificial flavin-binding protein.

44. A method of producing an artificial protein or its derivative, wherein functional molecules are screened from among the translation products of the artificial gene according to claim 30 or 31.

45. A fusion protein or its derivative wherein the artificial protein or its derivative according to any of claims 39-43 and a marker protein and/or a peptide tag are bound.

46. A transgenic non-human animal having the potential for expressing the artificial protein or its derivative according to any of claims 39-43.

47. A transgenic plant having the potential for expressing the artificial protein or its derivative according to any of claims 39-43.

48. A drug for the treatment of various diseases, wherein the artificial protein or its derivative according to any of claims 39-43 is contained as an effective component.

49. A drug for the diagnosis of various diseases, wherein the artificial protein or its derivative according to any of claims 39-43 is contained as an effective component.

50. An artificial biological tissue wherein the artificial protein or its derivative according to any of claims 39-43 is contained as an effective component.

51. An artificial protein polymer material wherein the artificial protein or its derivative according to any of claims 39-43 is contained as an effective component.

14. A method of producing a multifunctional base sequence having two or more functions wherein a base sequence is selected, from among all the combinations of base sequences encoding an amino acid sequence having a given function, which has a function same as or different from the given function in a reading frame different from that of the amino acid sequence having the given function.

15. The method of producing a multifunctional base sequence having two or more functions according to claim 14, wherein the base sequence, which has a function same as or different from the given function, is selected by the computational science approach.

16. The method of producing a multifunctional base sequence having two or more functions according to claim 15, wherein the computational science approach is an approach to make assessments and selections based on the scores obtained by using a biological function prediction program.

17. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-16, wherein the base sequence is a double-stranded base sequence.

18. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-17, wherein the base sequence is DNA.

19. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-17, wherein the base sequence is RNA.

20. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-19, wherein the base sequence is a linear base sequence.

21. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-20, wherein all three reading frames emerging from the one-by-one frameshifts of the reading frames in the base sequence are devoid of termination codons.

22. The method of producing a multifunctional base sequence having two or more functions according to any of claims 17-21, wherein all six reading frames of the base sequence are devoid of termination codons.

23. The method of producing a multifunctional base sequence having two or more functions according to claim 21 or 22, wherein termination codons do not emerge at the junction points arising from the polymerization of the multifunctional base sequence.

24. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-23, wherein the two or more functions are two or more biological functions.

25. The method of producing a multifunctional base sequence having two or more functions according to claim 24, wherein the biological functions are:

function of easily forming secondary structures;

antigen function to induce neutralizing antibodies;

function to activate immunity;

function to promote or suppress cell proliferation;

function to specifically recognize cancer cells;

protein transduction function;

cell-death-inducing function;

function to present residues that determine antigens;

metal-binding function;

coenzyme-binding function;

function to activate catalysts;

function to activate fluorescence signal;

function to bind to a specific receptor and to activate the receptor;

function to bind to a specific factor involved in signal transduction and to modulate the action of the factor;

function to specifically recognize biopolymers;

cell adhesion function;

function to localize proteins to the cell exterior;

function to target at a specific intracellular organelle;

function to be embedded in the cell membrane;

function to form amyloid fibers;

function to form fibrous proteins;

function to form a protein gel;

function to form a protein film;

function to form a single molecular membrane;

self-aggregation function;

function to form particles;

or function to assist the formation of higher-order structure of other proteins.

26. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-25, wherein the base sequence comprises 15-500 bases or base pairs.

27. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-26, wherein modification is further performed for polymerization of the multifunctional base sequence.

28. The method of producing a multifunctional base sequence having two or more functions according to any of claims 14-27, wherein a natural base sequence is further bound thereto.

29. A multifunctional base sequence having two or more functions which can be produced by the method of producing a multifunctional base sequence having two or more functions according to any of claims 14-28.