Site-specific labeling of affinity tags in fusion proteins

• US 9,164,099 B2
• Filed: 05/08/2013
• Issued: 10/20/2015
• Est. Priority Date: 09/12/2002
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Gee, Kyle, Rukavishnikov, Aleksey, Haugland, Richard, Leung, Wai-Yee, Patton, Wayne, Diwu, Zhenjun, Hart Kerndt, Courtenay
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/890,137
• Publication Number:
  US 20140038856A1
• Time in Patent Office: 895 Days
• US Class Current: 1/1
• 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 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: C07F 5/022 without C-boron linkages
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• 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

1. A compound having the structure: