Methods for the detection, analysis and isolation of nascent proteins

• US 20030092031A1
• Filed: 06/18/2002
• Published: 05/15/2003
• Est. Priority Date: 08/25/1999
• Status: Active Grant

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Olejnik, Jerzy, Gite, Sadanand, Rothschild, Kenneth J.
• Granted Patent: US 7,897,335 B2
• Time in Patent Office: Days
• US Class Current: 435/6.13
• 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 15/67 General methods for enhancing the expression
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.

Claims

11. A gel-free method, comprising:

a) providing i) a sample comprising template nucleic acid, ii) primers capable of introducing sequences encoding an N-terminal marker and a C-terminal marker into said template nucleic acid so as to create modified template, iii) misaminoacylated tRNA comprising a biotin marker, and iv) a translation system;

b) diluting at least a portion of said sample to create a plurality of diluted samples at least a portion of which contain some template;

c) amplifying said plurality of diluted samples with said primers under conditions such that said sequences encoding said N-terminal and C-terminal markers are introduced into at least some of said nucleic acid template such that modified template encoding a protein is produced as an amplification product;

d) introducing said modified template and misaminoacylated tRNA into said translation system under conditions such that a nascent protein is generated, said protein comprising at least said N-terminal marker;

e) isolating said nascent protein from said translation system by binding said nascent protein to a biotin-binding ligand; and

f) testing said nascent protein under gel-free conditions that permit the detection of a truncated protein.

1. A gel-free method, comprising:

a) providing i) a sample comprising template nucleic acid, ii) primers capable of introducing sequences encoding an N-terminal marker and a C-terminal marker into said template nucleic acid so as to create modified template, and iii) a translation system;

b) diluting at least a portion of said sample to create a plurality of diluted samples at least a portion of which contain some template;

c) amplifying said plurality of diluted samples with said primers under conditions such that said sequences encoding said N-terminal and C-terminal markers are introduced into at least some of said nucleic acid template such that modified template encoding a protein is produced as an amplification product;

d) introducing said modified template into said translation system under conditions such that a nascent protein is generated, said protein comprising at least said N-terminal marker; and

e) testing said nascent protein under gel-free conditions that permit the detection of a truncated protein.

2. The method of claim 1, wherein the diluting of step (b) is performed until between 0.1 and 0.9 of said diluted samples yield an amplification product after step (c).

3. The method of claim 1, wherein said testing of step (e) is performed on a solid support.

4. The method of claim 3, wherein said solid support comprises a microtiter plate.

5. 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.

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

7. The method of claim 6, 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.

8. The method of claim 6, 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.

9. The method of claim 6, wherein said cell-free translation system is incubated at a temperature of between about 25°

C. to about 45°

C.

10. The method of claim 6, wherein the cell-free translation system is a continuous flow or dialysis system.

12. The method of claim 11, wherein the diluting of step (b) is performed until between 0.1 and 0.9 of the diluted samples yield an amplification product.

13. The method of claim 11, wherein the tRNA molecule is aminoacylated by chemical or enzymatic misaminoacylation.

14. The method of claim 11, wherein said nascent protein detected is functionally active.

15. The method of claim 11, wherein said biotin-binding ligand is selected from the group consisting of avidin and streptavidin.

13-1. The method of claim 11, wherein two or more different misaminoacylated tRNAs are introduced into the translation system.