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Synthetic luciferase gene and protein

a luciferase gene and synthetic technology, applied in the direction of peptides, drug compositions, immunological disorders, etc., can solve the problems that current luciferases isolated from various organisms, including insects and marine organisms, are not necessarily optimized for expression or production, and achieve the effect of improving thermostability

Inactive Publication Date: 2014-03-27
MARKER GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about a new and improved luciferase gene that can be used to produce a stabilized, high-quality luciferase protein. This new protein has the ability to emit light of a longer wavelength and is more stable and expressible in mammalian cell systems compared to native luciferase. The method described involves inserting a nucleic acid molecule of the invention into a microorganism and growing it to produce the desired stable luciferase protein.

Problems solved by technology

Despite its utility as a reporter, current luciferases isolated from various organisms, including insects and marine organisms are not necessarily optimized for expression or production in systems that are of most interest to the medical community and experimental molecular biologists.

Method used

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  • Synthetic luciferase gene and protein
  • Synthetic luciferase gene and protein
  • Synthetic luciferase gene and protein

Examples

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example 1

Synthetic Construction of the COS Luciferase Gene

[0071]The synthetic COS luciferase gene, SEQ ID NO:3, was assembled from synthetic oligonucleotides and / or PCR products. The fragment was cloned into pMK (kanR) using KpnI and SacI restriction sites. The plasmid DNA was purified (Pure Yield™ Plasmid Midiprep, Promega) from transformed bacteria and concentration determined by UV spectroscopy. The final construct was verified by sequencing. The sequence congruence within the used restriction sites was 100%.

example 2

Subcloning of the COS Luciferase Gene into the pCMV and pSV40 Vectors

[0072]The synthetic COS luciferase assembled in Example 1 was excised from pMK cloning vector using flanking XhoI and NotI restriction enzymes (Fast Digest, Fermentas). The excised fragment was gel-purified (GenElute Gel Extraction Kit, Sigma) and quantitated using MassRuler™ DNA Ladder Mix (Fermentas). The excised gene was subcloned into both pCMV and pSV40 Mammalian Expression Vectors using corresponding XhoI and NotI restriction sites. The completed pCMV construct was named pDC57. The completed pSV40 construct was named pDC99.

example 3

Subcloning of the COS Luciferase Gene into the pNosdc Binary Vector for Expression in Plants

[0073]The synthetic COS luciferase assembled in Example 1 was amplified using the Polymerase Chain reaction. Amplification was performed with primers including XmaI and SacI restriction sites. The ends of the amplified fragment were cut with XmaI and SacI restriction enzymes (New England Biolabs) and the fragment was gel-purified (GenElute Gel Extraction Kit, Sigma) and quantitated using MassRuler™ DNA Ladder Mix (Fermentas). The amplified fragment was subcloned into the pNosdc binary vector for transformation of plants via Agrobacterium tumefaciens. The completed construct was named pNosdcCOS.

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Abstract

A codon optimized and stabilized luciferase gene and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase. Assays using this new enzyme for measuring various biological metabolic functions are described.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a novel codon optimized and stabilized luciferase gene (COS luciferase) and protein and uses thereof. The invention further relates to production of a stabilized luciferase protein using such a COS gene and methods of analysis of cells using the luciferase gene and protein.BACKGROUND OF THE INVENTION[0002]Bioluminescence in certain organisms via the reaction of luciferin and luciferase is well known in the art. The use of the luciferase enzyme has become highly valuable as a genetic marker gene due to the convenience, sensitivity and linear range of the luminescence assay. Luciferase has been used in many experimental biological systems in both prokaryotic and eukaryotic cell culture, transgenic plants and animals, as well as cell-free expression systems.[0003]For example, Japanese Firefly Luciola cruciata luciferase expression can be monitored as a genetic marker in cell extracts when mixed with substrates (D-luciferin, M...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/02C12Q1/66
CPCC12Q1/66C12N9/0069C07K2317/33C07K2317/34C07K2317/55C07K2317/76C07K2317/92C07K16/2866A61P1/00A61P1/04A61P17/06A61P19/02A61P29/00A61P37/00A61P37/02
Inventor COLEMAN, DANIEL J.NALEWAY, JOHN J.COOK, GABRIELE M.JIANG, YING
Owner MARKER GENE TECH
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