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Firefly luciferase and gene thereof, and process for production of firefly luciferase

一种荧光素酶基因、荧光素酶的技术，应用在萤火虫荧光素酶、其基因和萤火虫荧光素酶的制造法领域，能够解决荧光素酶反应障碍、突变荧光素酶取得困难、无法广泛用试剂等问题

Inactive Publication Date: 2012-07-18
KIKKOMAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has the following disadvantages: it cannot be widely used for various purposes and reagents due to various restrictions on the composition of the reagents; in addition, in most cases, the addition of salts tends to cause a certain degree of reaction disturbance of the luciferase reaction
That is, even if multiple mutations considered to be useful are introduced separately, it is difficult to say that these preferred properties are simply superimposed or synergistically endowed. It becomes more difficult to obtain Suzyme

Method used

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  • Firefly luciferase and gene thereof, and process for production of firefly luciferase
  • Firefly luciferase and gene thereof, and process for production of firefly luciferase
  • Firefly luciferase and gene thereof, and process for production of firefly luciferase

Examples

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Effect test

preparation example Construction

[0047] (Preparation of vectors and preparation of transformants)

[0048] The mutant firefly luciferase gene obtained as above is recombined into vectors such as bacteriophage, cosmid, or plasmids for transforming prokaryotic cells or eukaryotic cells by conventional methods, and the host is treated with the vector by conventional methods. Transform or transduce.

[0049] Any species can be used as the host, and microorganisms such as microorganisms are preferable. Specifically, microorganisms belonging to the genus Escherichia or the like can be used. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli K-12, JM109, DH5α, HB101, BL21 and the like.

[0050] Then, the obtained transformants or transduced hosts are screened for strains capable of producing firefly luciferase with desired mutations, so as to obtain strains capable of producing mutant firefly luciferase. In order to obtain purified novel recombinant DNA from the strain thus obt...

Embodiment 1

[0060] (Preparation of plasmid pET16b-BLU-Y)

[0061] A full-length primer (SEQ ID NO: 1) for amplifying the biotinylated luciferase structural gene was synthesized. Using the plasmid pHLf248 described in Japanese Patent No. 3466765 (Escherichia coli JM101 [pHLf248] containing this plasmid is deposited in the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology, designated as FERM BP-5081.) as a template, the above-mentioned The full-length DNA fragment was amplified by PCR using primers and commercially available M13-M4 primers (manufactured by Takara Bio, Inc.). The obtained DNA fragment was digested with NdeI and HindIII, subjected to agarose gel electrophoresis, and the DNA fragment was purified from a 1.9 kb band.

[0062] Next, the aforementioned DNA fragment was inserted into the plasmid vector pET16b (manufactured by Novagen) digested with NdeI and HindIII by a conventional method to prepare plasmid pET16b-BLU-Y.

[0063]...

Embodiment 2

[0065] (Preparation of a plasmid containing a mutant firefly luciferase gene)

[0066] A plasmid (pHLf344A) containing a gene encoding a luciferin in which the amino acid residue leucine at position 344 of the amino acid sequence of Heike luciferase was mutated to alanine (344A mutation) was prepared as follows enzyme.

[0067] First, PCR primers (pHLf344A F-344A (SEQ ID NO: 2), pHLf344A R-344A (SEQ ID NO: 3)) designed by replacing the 344th amino acid residue from leucine to alanine were synthesized. These primers were used to carry out PCR amplification with pET16b-BLU-Y as a template (reaction steps and conditions followed conventional methods). The amplified PCR reaction solution was digested with DpnI, and the end of the PCR product was phosphorylated by kinase treatment. Next, using the Ligation Convenience Kit (manufactured by Nippon Gene Co., Ltd.), the reaction product of the PCR product treated with DpnI and kinase was self-ligated (self-ligation) to obtain pHLf344...

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Abstract

Disclosed are: a firefly luciferase having excellent thermal stability and / or storage stability; and a process for producing the firefly luciferase. Specifically disclosed are: a firefly luciferase characterized by comprising an amino acid sequence that is produced by substituting an amino acid residue located at position-287 by an alanine residue or mutating an amino acid residue located at position-392 by an isoleucine residue in the amino acid sequencer for a Japanese firefly Luciola lateralis; and a gene for the firefly luciferase. The use of the gene enables the efficient production of a firefly luciferase having improved stability. The above-mentioned mutation may be combined with a mutation produced by substituting an amino acid residue located at position-326 by a serine residue and / or a mutation produced by substituting an amino acid residue located at position-467 by an isoleucine residue in the same amino acid sequence, thereby producing a firefly luciferase having further improved stability.

Description

[0001] The present invention relates to the invention related to the commissioned research results of "Heisei 20 and 21, Science and Technology Promotion Organization, Advanced Measurement and Analysis Technology and Device Development Project". technical field [0002] The present invention relates to a mutant firefly luciferase, a mutant firefly luciferase gene, a novel recombinant DNA, and a method for producing a mutant firefly luciferase, specifically, firefly luciferase with improved stability, and its Gene and stability of firefly luciferase. Background technique [0003] Firefly luciferase is an enzyme that converts adenosine triphosphate (ATP), D-luciferin and oxygen into adenosine monophosphate (AMP), oxidizes luciferin and carbon dioxide in the presence of magnesium ions and oxygen and produces light. The luminescence principle of firefly luciferase can be used to determine the microenzyme reaction substrate with extremely high sensitivity. Therefore, firefly luc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N9/02C12N15/09
CPCC12N9/0069C12Y113/12007
Inventor 小玉侑加子吉原绘里子
Owner KIKKOMAN CORP
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