Detection of markers in nascent proteins

• US 6,919,179 B2
• Filed: 12/27/2001
• Issued: 07/19/2005
• 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:
  US10/034,736
• Publication Number:
  US 20030059785A1
• Time in Patent Office: 1,300 Days
• Field of Search: 435/6, 435/70.1, 435/71.1, 536/24.1, 536/25.32
• 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 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/582 with fluorescent label

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 mammals, and kits which may be used for the detection of diseases and disorders.

Claims

1. A method, comprising

a) providing i) tRNA, ii) a non-radioactive marker, and iii) a translation system;

b) aminoacylating said tRNA with said marker 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; and

d) binding said nascent protein to a surface.

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

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

4. The method of claim 3, wherein the cellular translation system is selected from the group consisting of tissue culture cells, primary cells, cells in vivo, isolated immortalized cells, human cells and combinations thereof.

5. The method of claim 3, wherein the cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, frog oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

6. The method of claim 1, wherein said surface is selected from the group consisting of a tissue culture plate surface, microtiter plate surface, surface of a bead, glass surface and polymer surface.

7. The method of claim 3, wherein the cell-free translation system is a continuous flow or dialysis system.

8. The method of claim 1, wherein the tRNA molecule is aminoacylated by chemical or enzymatic misaminoacylation.

9. The method of claim 1, wherein two or more different misaminoacylated tRNAs are introduced into the translation system.

10. The method of claim 1, wherein said nascent protein is functionally active.

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

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

13. A method, comprising

a) providing i) tRNA, ii) a non-radioactive marker, and iii) a translation system;

b) aminoacylating said tRNA with said marker 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) binding said nascent protein to a surface to create a bound nascent protein; and

e) detecting said bound nascent protein.

14. The method of claim 13, wherein the nascent protein detected is selected from recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate and lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic proteins and fragments and combinations thereof.

15. The method of claim 13, wherein said translation system comprises a cellular or cell-free translation system.

16. The method of claim 15, wherein the cellular translation system is selected from the group consisting of tissue culture cells, primary cells, cells in vivo, isolated immortalized cells, human cells and combinations thereof.

17. The method of claim 15, wherein the cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, frog oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

18. The method of claim 13, wherein said surface is selected from the group consisting of a tissue culture plate surface, microtiter plate surface, surface of a bead, glass surface and polymer surface.

19. The method of claim 15, wherein the cell-free translation system is a continuous flow or dialysis system.

20. The method of claim 13, wherein the tRNA molecule is aminoacylated by chemical or enzymatic misaminoacylation.

21. The method of claim 13, wherein two or more different misaminoacylated tRNAs are introduced into the translation system.

22. The method of claim 13, wherein said nascent protein detected is functionally active.

23. The method of claim 13, wherein said tRNA molecule is an initiator tRNA molecule.

24. The method of claim 13, wherein said tRNA molecule is a suppressor tRNA molecule.

25. A method, comprising

a) providing i) tRNA, ii) a fluorescent marker, and iii) a translation system;

b) aminoacylating said tRNA with said marker to create a misaminoacylated tRNA;

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

d) binding said fluorescent nascent protein to a surface to create a bound, fluorescent nascent protein; and

e) detecting said bound, fluorescent nascent protein.

26. The method of claim 25, wherein the nascent protein detected is selected from recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate and lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic proteins and fragments and combinations thereof.

27. The method of claim 25, wherein said translation system comprises a cellular or cell-free translation system.

28. The method of claim 27, wherein the cellular translation system is selected from the group consisting of tissue culture cells, primary cells, cells in vivo, isolated immortalized cells, human cells and combinations thereof.

29. The method of claim 27, wherein the cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, frog oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

30. The method of claim 25, wherein said surface is selected from the group consisting of a tissue culture plate surface, microtiter plate surface, surface of a bead, glass surface and polymer surface.

31. The method of claim 27, wherein the cell-free translation system is a continuous flow or dialysis system.

32. The method of claim 25, wherein the tRNA molecule is aminoacylated by chemical or enzymatic misaminoacylation.

33. The method of claim 25, wherein two or more different misaminoacylated tRNAs are introduced into the translation system.

34. The method of claim 25, wherein said nascent protein detected is functionally active.

35. The method of claim 25, wherein said tRNA molecule is an initiator tRNA molecule.

36. The method of claim 25, wherein said tRNA molecule is a suppressor tRNA molecule.