Detection of markers in nascent proteins

• US 7,211,394 B2
• Filed: 06/06/2005
• Issued: 05/01/2007
• 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:
  US11/145,781
• Publication Number:
  US 20060024704A1
• Time in Patent Office: 694 Days
• Field of Search: None
• 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) a composition comprising a nascent protein comprising an affinity marker and a photocleavable detectable label, and

ii) a surface comprising an acceptor molecule; and

b) interacting said affinity marker with said acceptor molecule so as to form a protein coated surface.

2. The method of claim 1, wherein said affinity marker is a selected from the group consisting of amino acids, amino acid derivatives, and amino acid analogs in which a coupling agent is attached.

3. The method of claim 2, wherein said coupling agent is selected from the group consisting of haptins, immunogenic molecules, biotin, biotin derivatives, and fragments and combinations of these molecules.

4. The method of claim 1, wherein said detectable label is selected from the group consisting of a ferromagnetic detectable label, paramagnetic detectable label, diamagnetic detectable label, luminescent detectable label, electrochemiluminescent detectable label, fluorescent detectable label, phosphorescent detectable label, and chromatic detectable label.

5. The method of claim 4, wherein said fluorescent detectable label is selected from the group consisting of dansyl fluorophores, cumarin derivatives, acidinium fluorophores, and benzopyrene fluorophores.

6. The method of claim 4, wherein said electrochemiluminescent detectable label is selected from the group consisting of ruthenium chelates and nitroxide amino acids.

7. A method, comprising:

a) providing

i) a composition comprising a nascent protein comprising an affinity marker and a photocleavable detectable label; and

ii) a surface comprising an acceptor molecule;

b) interacting said affinity marker with said acceptor molecule so as to form a nascent protein coated surface;

c) exposing said protein coated surface to electromagnetic radiation releasing said detectable label form said protein; and

d) detecting said cleaved detectable label.

8. The method of claim 7, wherein said affinity marker is a selected from the group consisting of amino acids, amino acid derivatives, and amino acid analogs in which a coupling agent is attached.

9. The method of claim 8, wherein said coupling agent is selected from the group consisting of haptins, immunogenic molecules, biotin, biotin derivatives, and fragments and combinations of these molecules.

10. The method of claim 7, wherein said detectable label is selected from the group consisting of a ferromagnetic detectable label, paramagnetic detectable label, diamagnetic detectable label, luminescent detectable label, electrochemiluminescent detectable label, fluorescent detectable label, phosphorescent detectable label, and chromatic detectable label.

11. The method of claim 10, wherein said fluorescent detectable label is selected from the group consisting of dansyl fluorophores, cumarin derivatives, acidinium fluorophores, and benzopyrene fluorophores.

12. The method of claim 10, wherein said electrochemiluminescent detectable label is selected from the group consisting of ruthenium chelates and nitroxide amino acids.