Site-specific labeling of affinity tags in fusion proteins

• US 20060141554A1
• Filed: 10/14/2004
• Published: 06/29/2006
• Est. Priority Date: 09/12/2002
• Status: Abandoned Application

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard, Patton, Wayne Forrest, Gee, Kyle Richard, Whitney, Scott, Hart, Courtenay Rae
• Application Number:
  US10/966,536
• Publication Number:
  US 20060141554A1
• Time in Patent Office: Days
• US Class Current: 435/40.500
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0039 Coumarin dyes
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07D 263/62 having two or more ring systems containing condensed 1,3-oxazole rings
• CPC Class Codes: C07D 311/12 substituted in position 3 and unsubstituted in position 7
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 311/18 substituted otherwise than in position 3 or 7
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07F 5/022 without C-boron linkages
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention provides methods and fluorescent compounds that facilitate detecting and labeling of a fusion protein by being capable of selectively binding to an affinity tag. The fluorescent compounds have the general formula A(B)n, wherein A is a fluorophore, B is a binding domain that is a charged chemical moiety, a protein or fragment thereof and n is an integer from 1-6 with the proviso that the protein or fragment thereof not be an antibody or generated from an antibody. The present invention provides specific fluorescent compounds and methods used to detect and label fusion proteins that contain a poly-histidine affinity tag. These compounds have the general formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain, m is an integer from 1 to 4 and n is an integer from 1 to 6. The acetic acid groups interact directly with the positively charged histidine residues of the affinity tag to effectively label and detect a fusion protein containing such an affinity tag when present in an acidic or neutral environment.

Claims

2. A staining solution comprising:

a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6; and

,

b) a buffer having a pH of about 7.0 to about 9.0

with the proviso that the binding domain does not comprise an antibody or fragment thereof.

4. The staining solution according to claim 2, wherein the metal ion is nickel or cobalt.

5. The staining solution according to claim 2, wherein the fluorescent compound is

12. A staining solution comprising

a) a fluorescent compound comprising nickel ions, wherein the fluorescent compound has the formula A(L)m(B)n wherein A is a fluorophore that is xanthene, coumarin, cyanine, acridine, anthracene, benzofuran, indole or borapolyazaindacene, L is a linker, B is an acetic acid binding domain that is NTA or BAPTA, m is an integer from 1 to 4 and n is an integer from 1 to 6;

and,

b) a buffer having a pH of about 7.0 to about 9.0 comprising about 20 mM phosphate;

with the proviso that the binding domain does not comprise an antibody or fragment thereof.

18. A method for detecting a poly-histidine affinity tag containing fusion protein in a sample, the method comprising:

i) immobilizing the sample on a solid or semi-solid matrix to prepare an immobilized sample;

ii) contacting the immobilized sample with a staining solution to prepare a stained sample, wherein the staining solution comprises

a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6; and

,

b) a buffer having a pH of about 7.0 to about 9.0;

iii) incubating the stained sample for a sufficient amount of time to allow the fluorescent compound to associate with the poly-histidine affinity tag to prepare an incubated sample;

iv) illuminating the incubated sample with a suitable light source to prepare an illuminated sample; and

v) observing the illuminated sample whereby the fusion protein is detected.

36. A kit for detecting an affinity tag containing fusion protein, wherein the kit comprises;

a staining solution comprising;

a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6; and

,

b) a buffer having a pH of about 7.0 to about 9.0;

with the proviso that the binding domain does not comprise an antibody or fragment thereof.

38. The kit according to claim 36, wherein the additional detection reagent is a total protein stain.

42. The kit according to claim 35, wherein the fluorescent compound is

42-1. A fluorescent compound having the chemical structure: