Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-06-26
2004-11-02
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S252330, C435S254110, C435S320100, C435S325000, C536S023200
Reexamination Certificate
active
06812012
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel luciferase having resistance to a surfactant and a method for measuring intracellular ATP using the same.
BACKGROUND ART
Intracellular ATP is routinely measured for determining the presence of cells in a sample or the number of cells in the fields of food sanitation, biology, clinical examinations, medical science, ultrapure water, and environmental science. A general method for measuring intracellular ATP comprises the steps of adding an ATP extraction reagent containing a surfactant as an effective component to a sample containing cells, extracting intracellular ATP, adding a luminescence reagent containing luciferase into the sample, and then measuring the total amount of light emitted.
Luciferase is an enzyme that catalyzes luminescence reaction of luciferin, which is a substrate, in the presence of ATP and magnesium ion. Luciferase used in a method for measuring intracellular ATP includes those derived from firefly species, such as GENJI firefly (
Luciola cruciata
), HEIKE firefly (
Luciola lateralis
), North American firefly and Russian firefly, etc.
Intracellular ATP can be extracted by adding an ATP extraction reagent to a sample containing cells and then stirring the sample.
To make full use of the capabilities of the extraction reagent, preferably the reaction agent is added so that the concentration of a surfactant becomes 0.05% or more of the mixture of the sample and the extraction reagent. However, a condition where the concentration of the surfactant is 0.05% or more, this inhibits significantly the enzyme reaction in the process of measuring ATP concentration by bioluminescence. Thus the sensitivity and accuracy of measurement are largely impaired. This is because a surfactant at such a high concentration lowers luciferase activity.
For example, North American firefly luciferase activity decreases to about 20% in the presence of 0.1% enzalkonium chloride (See Table 1).
On the other hand, inhibition of the bioluminescent reaction can be reduced with a lower concentration of surfactant. However, in this case the extraction efficiency for ATP would be insufficient.
A method wherein cyclodextrin or its derivative is used is a known method for suppressing the inhibition of luminescence reaction by a surfactant (Japanese Patent Application Laid-Open No. 6-504200).
Among methods for measuring intracellular ATP wherein intracellular ATP is extracted by allowing a sample to contact with a surfactant and subsequently ATP is measured by luciferin-luciferase bioluminescent reaction method, a method for measuring intracellular ATP characterized by the application of the bioluminescent reaction method after allowing a sample, from which ATP is extracted, to contact with cyclodextrin (Japanese Patent Application Laid-Open Publication No. 7-203995) is also known.
There has been no attempt so far to suppress the inhibition of bioluminescent reaction due to a surfactant focusing on luciferase.
The purpose of the invention is to provide a novel luciferase having anti-surfactant resistance, whose activity is not impaired by the presence of a surfactant at a high concentration. The other purpose of the invention is to provide a method, comprising the steps of extracting intracellular ATP using a surfactant and measuring intracellular ATP by bioluminescent reaction using a luciferase, which can lower the inhibition of bioluminescent reaction due to a surfactant without a decrease in efficiency in extracting intracellular ATP.
In the context of this specification, the term “suppress” is used to describe significant reduction of the inhibition of the luminescence reaction by a surfactant and the complete elimination of this inhibition.
DISCLOSURE OF THE INVENTION
The present invention relates to a luciferase having anti-surfactant resistance.
The luciferase having resistance to a surfactant includes a luciferase, wherein an amino acid at the 490-position, or an amino acid corresponding to the amino acid at 490-position of GENJI firefly or HEIKE firefly is substituted by an amino acid other than amino acid, e.g., lysine in the amino acid sequence of a wild-type firefly luciferase.
Further, the luciferase having resistance to a surfactant includes a polypeptide consisting of (a) or (b):
(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO-4,
(b) A polypeptide comprising additions, deletions, or substitutions of one or more of amino acids in the polypeptide of (a), and having luciferase activity resistant to a surfactant, or
a polypeptide consisting of (a) or (b):
(a) A protein consisting of an amino acid sequence shown in SEQ ID NO:6,
(b) A protein comprising additions, deletions, or substitutions of one or more of amino acids in the polypeptide of (a), and having luciferase activity resistant to a surfactant.
Further, the present invention relates to a luceferase gene encoding the luciferase having resistance to a surfactant.
Furthermore, the present invention relates to a recombinant vector containing the luceiferase gene encoding the luciferase having resistance to a surfactant.
The present invention also relates to a transformant containing the recombinant vector.
In addition, the present invention relates to a method for producing the luciferase, comprising the steps of culturing the recombinant in a medium, and collecting luciferase with resistance to a surfactant from the culture product.
Moreover the present invention relates to a method for measuring intracellular ATP, comprising the steps of a first step wherein ATP is extracted in the presence of a surfactant from cells in a sample; a second step wherein a luminescence reagent containing luciferase is added to the extracted ATP solution so as to cause light emission; and a third step wherein the light emission is measured, and characterized in that luciferase having resistance to a surfactant is used.
This specification encompasses the description and/or drawings given in Japanese Patent Application No. H09-361022.
REFERENCES:
patent: 5229285 (1993-07-01), Kajiyama et al.
patent: 6074859 (2000-06-01), Hirokawa et al.
patent: 0 524 448 (1993-01-01), None
patent: 2-17189 (1990-07-01), None
patent: 5-244942 (1993-09-01), None
patent: 6-504200 (1994-05-01), None
patent: 7-203995 (1995-08-01), None
patent: WO 92/12253 (1992-07-01), None
patent: WO 96/07759 (1996-03-01), None
Tatsumi et al. (1992) Biochimica & Biophysica Acta, vol. 1131, pp. 161-165.*
Simpson et al. (1991) Journal of Bioluminescence and Chemiluminescence, vol. 6, pp. 97-106.*
W. J. Simpson, et al., The Journal of Bioluminescence and Chemiluminescence, vol. 6, No. 2, pp. 97-106, “The Effect of Detergents on Firefly Luciferase Reactions”, 1991.
H. Tatsumi, et al., Biochimica et Biophysica Acta, vol. 1131, pp. 161-165, “Molecular Cloning and Expression inEscherichia coliof a cDNA Clone Encoding Luciferase of a Firefly, Luciola Lateralis”, 1992.
T. Masuda, et al., Gene, vol. 77, pp. 265-270, “Cloning and Sequence Analysis of cDNA for Lucifirase of a Japanese Firefly,Luciola Cruciata”, 1988.
Hattori Noriaki
Murakami Seiji
Kikkoman Corporation
Slobodyansky Elizabeth
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