Methods for the detection and isolation of proteins

• US 6,210,941 B1
• Filed: 04/12/1999
• Issued: 04/03/2001
• Est. Priority Date: 06/27/1997
• Status: Expired due to Term

• Current Assignee: 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:
  US09/290,325
• Time in Patent Office: 722 Days
• Field of Search: 435/183, 435/194, 435/212, 536/24.1
• US Class Current: 435/183
• 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 mammals, and kits which may be used for the detection of diseases and disorders.

Claims

1. A kit, comprising:

a) a first containing means containing at least one component of a protein synthesis system;

b) a second containing means containing a misaminoacylated tRNA, wherein said tRNA is misaminoacylated with a non-radioactive marker having a detectable electromagnetic spectral property.

2. The kit of claim 1, wherein said misaminoacylated tRNA comprises an initiator tRNA.

3. The kit of claim 1, wherein said misaminoacylated tRNA comprises a suppressor tRNA.

4. The kit of claim 1, wherein said marker is a photocleavable marker.

5. The kit of claim 1, wherein said marker is magnetic.

6. The kit of claim 1, wherein said marker has a net electric charge.

7. The kit of claim 1, wherein said component of said protein synthesis system comprises ribosomes.

8. The kit of claim 1, wherein said component of said protein synthesis system is selected from the group consisting of reducing agents, nucleoside triphosphates, initiation factors, elongation factors and termination factors.

9. A kit, comprising:

a) a first containing means containing at least one component of a protein synthesis system;

b) a second containing means containing a misaminoacylated tRNA, wherein said misaminoacylated tRNA comprises a photocleavable marker.

10. The kit of claim 9, wherein said misaminoacylated tRNA comprises an initiator tRNA.

11. The kit of claim 9, wherein said misaminoacylated tRNA comprises a suppressor tRNA.

12. The kit of claim 9, wherein said component of said protein synthesis system comprises ribosomes.

13. The kit of claim 9, wherein said component of said protein synthesis system is selected from the group consisting of reducing agents, nucleoside triphosphates, initiation factors, elongation factors and termination factors.

14. A kit, comprising:

a) a first containing means containing at least one component of a cellular or cell-free protein translation system;

b) a second containing means containing a misaminoacylated tRNA, wherein said misaminoacylated tRNA comprises a photocleavable marker.

15. The kit of claim 14, wherein said misaminoacylated tRNA comprises an initiator tRNA.

16. The kit of claim 14, wherein said misaminoacylated tRNA comprises a suppressor tRNA.

17. The kit of claim 14, wherein said component comprises ribosomes.

18. The kit of claim 14, wherein said component of said protein synthesis system is selected from the group consisting of reducing agents, nucleoside triphosphates, initiation factors, elongation factors and termination factors.