Electrophoresis of nascent proteins

• US 6,344,320 B1
• Filed: 05/07/1999
• Issued: 02/05/2002
• Est. Priority Date: 05/11/1994
• Status: Expired due to Fees

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth J., Sonar, Sanjay M.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/307,579
• Time in Patent Office: 1,005 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.13
• 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 41/0042 Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
• CPC Class Codes: C07C 205/45 the carbon skeleton being further substituted by at least one doubly—bound oxygen atom, not being part of a —CHO group
• CPC Class Codes: C07C 205/57 having nitro groups and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The invention is directed to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent protein from other components of the translation system. The invention also comprises proteins prepared using misaminoacylated tRNAs which can be utilized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other maninials, and kits which may be used for the detection of diseases and disorders.

Claims

1. A method of detecting a marker incorporated into a nascent protein by exposure to an electric field, comprising

a) providing i) tRNA, ii) a marker, iii) an interactive molecule, and iv) a translation system;

b) aminoacylating said tRNA with said marker containing an alpha-amino acid to create a misaminoacylated tRNA;

c) introducing said misaminoacylated tRNA to said translation system under conditions such that said marker is incorporated into a nascent protein;

d) adding said interactive molecule to said nascent protein to create a mixture; and

e) exposing said mixture to an electric field under conditions such that said marker is detected.

2. The method of claim 1, wherein said interactive molecule contains one or more acceptor groups.

3. The method of claim 2, wherein said acceptor group of said interactive molecule binds to said marker of said nascent protein.

4. The method of claim 1, wherein said nascent protein is selected from the group consisting of recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate binding proteins, lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic derived proteins and fragments and combinations thereof.

5. The method of claim 1, wherein said translation system comprises a cellular or cell-free translation system.

6. The method of claim 5, wherein said cellular translation system is selected from the group consisting of tissue culture cells, isolated primary cells, isolated immortalized cells, isolated human cells and combinations thereof.

7. The method of claim 5, wherein said cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

8. The method of claim 1, wherein said conditions for said translation system comprise a temperature of between about 25°

C. to about 45°

C.

9. The method of claim 5, wherein said cell-free translation system is a continuous flow system.

10. The method of claim 1, wherein said tRNA molecule is aminoacylated by chemical misaminoacylation.

11. The method of claim 1, wherein said marker comprises a detectable label.

12. The method of claim 11, wherein said label has a detectable electromagnetic spectral property.

13. The method of claim 11, wherein said detectable label is selected from the group consisting of ferromagnetic moieties, paramagnetic moieties, diamagnetic moieties, fluorescent moieties, phosphorescent moieties, luminescent moieties, electrochemiluminescent moieties, chromatic moieties, moieties having a detectable electron spin resonance, electrical capacitance, dielectric constant and electrical conductivity, and combinations thereof.

14. The method of claim 1, wherein said tRNA molecule is an initiator tRNA molecule.

15. The method of claim 1, wherein said tRNA molecule is a suppressor tRNA molecule.

16. The method of claim 1, wherein said exposing to an electric field of step (e) comprises subjecting said mixture to electrophoresis.

17. The method of claim 16, wherein said electrophoresis comprises gel electrophoresis.

18. The method of claim 16, wherein said electrophoresis comprises capillary electrophoresis.

19. A method of detecting a marker incorporated into a nascent protein by electrophoresis, comprising

a) providing i) tRNA, ii) a marker, iii) an interactive molecule, and iv) a translation system;

b) aminoacylating said tRNA with said marker containing an alpha-amino acid to create a misaminoacylated tRNA;

c) introducing said misaminoacylated tRNA to said translation system under conditions such that said marker is incorporated into a nascent protein;

d) adding said interactive molecule to said nascent protein to create a mixture; and

e) subjecting said mixture to electrophoresis under conditions such that said marker is detected.

20. The method of claim 19, wherein said interactive molecule contains one or more acceptor groups.

21. The method of claim 20, wherein said acceptor group of said interactive molecule binds to said marker of said nascent protein.

22. The method of claim 19, wherein said nascent protein is selected from the group consisting of recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate binding proteins, lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic derived proteins and fragments and combinations thereof.

23. The method of claim 19, wherein said translation system comprises a cellular or cell-free translation system.

24. The method of claim 23, wherein said cellular translation system is selected from the group consisting of tissue culture cells, isolated primary cells, isolated immortalized cells, isolated human cells and combinations thereof.

25. The method of claim 23, wherein said cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

26. The method of claim 19, wherein said conditions for said translation system comprise a temperature of between about 25°

C. to about 45°

C.

27. The method of claim 23, wherein said cell-free translation system is a continuous flow system.

28. The method of claim 19, wherein said tRNA molecule is aminoacylated by chemical misaminoacylation.

29. The method of claim 19, wherein said marker comprises a detectable label.

30. The method of claim 29, wherein said label has a detectable electromagnetic spectral property.

31. The method of claim 29, wherein said detectable label is selected from the group consisting of ferromagnetic moieties, paramagnetic moieties, diamagnetic moieties, fluorescent moieties, phosphorescent moieties, luminescent moieties, electrochemiluminescent moieties, chromatic moieties, moieties having a detectable electron spin resonance, electrical capacitance, dielectric constant and electrical conductivity, and combinations thereof.

32. The method of claim 19, wherein said marker comprises a photocleavable marker.

33. The method of claim 19, wherein said tRNA molecule is an initiator tRNA molecule.

34. The method of claim 19, wherein said tRNA molecule is a suppressor tRNA molecule.

35. The method of claim 19, wherein said electrophoresis comprises gel electrophoresis.

36. The method of claim 19, wherein said electrophoresis comprises capillary electrophoresis.

37. A method of detecting a marker incorporated into a nascent protein by chromatography, comprising

a) providing i) tRNA, ii) a marker, iii) an interactive molecule, and iv) a translation system;

b) aminoacylating said tRNA with said marker containing an alpha-amino acid to create a misaminoacylated tRNA;

c) introducing said misaminoacylated tRNA to said translation system under conditions such that said marker is incorporated into a nascent protein;

d) adding said interactive molecule to said nascent protein to create a mixture; and

e) subjecting said mixture to chromatography under conditions such that said marker is detected.

38. The method of claim 37, wherein said chromatography comprises high-pressure liquid chromatography.

39. The method of claim 37, wherein said chromatography comprises affinity chromatography.

40. The method of claim 37, wherein said chromatography comprises ion exchange chromatography.

41. The method of claim 37, wherein said interactive molecule contains one or more acceptor groups.

42. The method of claim 41, wherein said acceptor group of said interactive molecule binds to said marker of said nascent protein.

43. The method of claim 37, wherein said nascent protein is selected from the group consisting of recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate binding proteins, lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic derived proteins and fragments and combinations thereof.

44. The method of claim 37, wherein said translation system comprises a cellular or cell-free translation system.

45. The method of claim 44, wherein said cellular translation system is selected from the group consisting of tissue culture cells, isolated primary cells, isolated immortalized cells, isolated human cells and combinations thereof.

46. The method of claim 44, wherein said cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

47. The method of claim 37, wherein said conditions for said translation system comprise a temperature of between about 25°

C. to about 45°

C.

48. The method of claim 44, wherein said cell-free translation system is a continuous flow system.

49. The method of claim 37, wherein said tRNA molecule is aminoacylated by chemical misaminoacylation.

50. The method of claim 37, wherein said marker comprises a detectable label.