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Luminescent markers

a technology of luminescent markers and fluorescent dyes, applied in the field of luminescent proteins and nucleic acids, can solve the problems of generating an acceptable signal-to-noise ratio and the use of luminescent markers, and achieve the effect of enhancing luminescence and thermostability

Inactive Publication Date: 2010-12-30
HOGUE CHRISTOPHER +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004]Applicants have produced an oncomodulin mutant that has unexpected advantages including but not limited to enhanced luminescence and thermostability.
[0005]Therefore, the invention provides a protein comprising oncomodulin in which one or more salt bridge and / or hydrogen bonding network has been introduced to provide greater stability when compared to a native oncomodulin or CDOM33.

Problems solved by technology

However, a significant limitation to the use of luminescent markers is generating an acceptable signal-to-noise ratio.

Method used

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  • Luminescent markers
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  • Luminescent markers

Examples

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example 1

Materials and Methods

[0145]ptacTBF Construct

[0146]TBF is a variant of oncomodulin in which the CD loop has been modified to bind terbium (III) as well as calcium (II) and an extended C-terminus for greater stability. The gene was synthesized by Operon Technologies, Inc. CA and designed so the gene would be flanked by EcoR I Hind III restriction sites for excise into other plasmids.

[0147]The gene was cut from the vector supplied by Operon Technologies and inserted into the pKK223-3 plasmid (Amersham Pharmacia Biotech) to take advantage of its tac promoter and the strong rrnB ribosomal terminator which stabilizes the plasmid by inhibiting read-through transcription initiated by the tac promoter. To do this, the vector containing the TBF gene and the pKK223-3 plasmid were both treated with the EcoR I and Hind III restriction enzymes (MBI Fermentas) at 37° C. for 2 hours and then run on a 1% agarose gel in TBE buffer to separate the fragments. The DNA was extracted from the gel using Ul...

example 2

[0185]pTTE Construct

[0186]The pTTE construct was designed to insert the protein sequence recognized by the TEV protease between the TBF and EGFP proteins. This was accomplished by designing a primer containing the code for the TEV recognition sequence and glycines flanked on either side to ensure accessibility of the enzyme to cleave (FIG. 11). As with the pTBFEGFP construct, an amino aid linker, threonine was needed to ensure transcription remained in frame through to EGFP. The coding strand primer was the same used for designing the pTBFEGFP construct as this plasmid was used as the template. The anticoding strand was designed as follows, 5′-CGCGCCATGGTaccgccaccgccACCCTGAAAATACAAATTCTCgccaccgccaccGGATT CAGCTACCATTTCTTGGAACTCATCAGCTCCAATCTTACC-3′ (Nco I site is in bold, codes for glycine are in lower case and the code for the TEV recognition sequence is bold and underlined) (SEQ ID NO. 11).

[0187]Amplification of the gene, restriction digest of the gene and the vector, ligation and ...

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Abstract

The invention relates to luminescent proteins, nucleic acids encoding same, compositions and combinations comprising the proteins, and methods using the proteins, nucleic acids, compositions and combinations. In particular, a luminescent protein is provided comprising oncomodulin in which a salt bridge has been introduced to provide greater stability. The protein may be used as a luminescent marker in, for example, luminescent items, immunoassays, and fluorescent energy transfer assays.

Description

[0001]This application is a Divisional of U.S. Ser. No. 10 / 961,645, filed 8 Oct. 2004, which is a Continuation of PCT / CA2003 / 000546, filed 11 Apr. 2003, which claims benefit of U.S. Ser. No. 60 / 372,382, filed 11 Apr. 2002 and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.FIELD OF THE INVENTION[0002]The invention relates to luminescent proteins, nucleic acids encoding same, compositions, complexes, and combinations comprising the proteins, and methods using the proteins, nucleic acids, complexes, compositions and combinations.BACKGROUND OF THE INVENTION[0003]Luminescent (including fluorescent and phosphorescent) markers have a wide variety of applications in science, medicine and engineering. In many cases, these markers provide competitive replacements for radiolabels, chromogens, radiation-dense dyes, etc. However, a significant limitation to the use of luminescent markers ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K49/00C07K14/00C07H21/04C07K16/00C12N15/63C12N5/10C12P21/06G01N21/76G01N33/567C07K14/435C07K14/47C12N15/12G01N33/533G01N33/542G01N33/58
CPCC07K14/43595C07K14/4728G01N33/582G01N33/533G01N33/542C07K2319/00
Inventor HOGUE, CHRISTOPHERSROKA, SUSANNA
Owner HOGUE CHRISTOPHER
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