JP2011188787A - Firefly luciferase, gene thereof, and method for producing firefly luciferase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a firefly luciferase having an excellent amount of luminescence, and to provide a method for producing the same. <P>SOLUTION: The firefly luciferase includes an amino acid sequence thereof. In the amino acid sequence, an amino acid corresponding to the 344th position of Luciola lateralis luciferase is mutated into alanine; the amino acid corresponding to the 287th position thereof is mutated into alanine; the amino acid corresponding to the 326th position thereof is mutated into serine; the amino acid corresponding to the 467th position thereof is mutated into isoleucine; and the amino acid corresponding to the 75th position thereof is mutated into alanine. The firefly luciferase having the improved amount of luminescence can be produced by utilizing the gene. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  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, a firefly luciferase with improved luminescence, a gene thereof, and a luminescence The present invention relates to a method for producing firefly luciferase.

  Firefly luciferase is an enzyme that converts adenosine triphosphate (ATP), D-luciferin and oxygen into adenosine monophosphate (AMP), oxyluciferin and carbon dioxide in the presence of magnesium ions and oxygen to produce light. It is. By applying the luminescence principle of firefly luciferase, a trace amount of enzyme reaction substrate can be measured with extremely high sensitivity. Therefore, for example, firefly luciferase is highly sensitive using, for example, detection of microorganisms in foods and drinks using ATP as an indicator, determination of food residues and dirt attached to fingers and instruments, or various antibody techniques and gene amplification techniques. Widely used in measurement methods.

However, in general, beetle luciferases such as firefly luciferase are unstable to heat and thus have a drawback of being easily inactivated when stored as a reagent. In general, the beetle luciferase has a problem that it is difficult to perform high-sensitivity measurement by a long-time reaction because the amount of luminescence rapidly decreases after the amount of luminescence reaches a peak immediately after the reaction. For this reason, attempts have been made to overcome such drawbacks and obtain a luciferase having good luminescence persistence, stability or high luminescence, and more desirably a luciferase having a plurality of excellent points.
One of the attempts is to devise a blending composition in which a salt or the like is added to the measurement reagent to ensure a certain degree of luminescence persistence, storage stability, and a high light emission amount. However, this method is not widely applicable to various uses / reagents, each of which has restrictions on the reagent composition, and in many cases, addition of salts causes some kind of reaction hindrance to the luciferase reaction. It has the disadvantage that it tends to be.

  One of the more preferable approaches that have been attempted in addition to devising the reagent composition is the search for mutant luciferases having favorable properties. In that attempt, North American firefly luciferase in which the amino acid at position 342 was mutated to alanine was obtained, and it has been reported that the emission persistence of this firefly luciferase is improved (for example, non-patent literature) 1). Further, the applicant obtains a mutant of Heikebotaruluciferase corresponding to the mutant of the amino acid at position 342, that is, Heikebotaruluciferase in which the amino acid at position 344 (leucine) is mutated to alanine (hereinafter referred to as 344A luciferase). It has been confirmed that the luminescence persistence is also improved in the mutant firefly luciferase. However, on the other hand, the stability of this mutant firefly luciferase is much lower than before the mutation was introduced.

Separately, the applicant has found a firefly luciferase with improved stability, including a mutation in which the amino acid at position 287 of Heike firefly luciferase is mutated to alanine and a mutation in which the amino acid at position 326 is mutated to serine. Based on this finding, in the 344A luciferase, the amino acid at position 287 was mutated to alanine, the amino acid at position 326 was mutated to serine, and the amino acid at position 467 was mutated to isoleucine. BLU-Y-A3T) has been newly obtained, and it has been confirmed that this mutant luciferase has excellent properties having both luminescence persistence and stability.
However, even with BLU-Y-A3T, which has improved industrially useful properties such as luminescence persistence and stability, there is a problem that the amount of luminescence is very low compared to that before mutation introduction. That is, in order to suitably apply the mutant firefly luciferase (BLU-Y-A3T) to high-sensitivity measurement and the like, it is desired to solve the problem of low light emission.

  Thus, in the search for mutant luciferases having favorable properties, even when a mutation that improves one property is found, the mutation often worsens another property. That is, even if a plurality of mutations that are considered to be useful individually are introduced, it is not simply said that these preferable properties are imparted additively or synergistically. The acquisition of mutant luciferase having a plurality of practically preferable properties such as properties, stability, and luminescence amount is made more difficult.

Biochemistry 2003, 42, p10429-10436

  An object of the present invention is to provide a firefly luciferase having preferable properties in all aspects of luminescence persistence, stability and luminescence amount by introducing a mutation into a specific base in the firefly luciferase gene sequence.

As a result of intensive studies to solve the above problems, the present inventor combined a mutation that replaces the amino acid at position 75 with alanine (hereinafter referred to as 75A mutation) to Heikebotaruluciferase (BLU-Y-A3T). It was found that the mutant firefly luciferase (BLU-Y-A3T-1) has desirable properties in all aspects of luminescence persistence, stability, and luminescence amount. Furthermore, for BLU-Y-A3T-1, a mutation that replaces the amino acid at position 293 with methionine (hereinafter referred to as 293M), a mutation that replaces the amino acid at position 351 with valine (hereinafter referred to as 351V), or position 172 Mutation for substituting the amino acid at 359 with glutamic acid (hereinafter referred to as 172E) and amino acid at position 259 with isoleucine (hereinafter referred to as 259I), or mutation for replacing amino acid at position 359 with valine (hereinafter referred to as 359V) and 490 Mutant firefly luciferase (in order BLU-Y-A3T-2, BLU-Y-A3T-3, BLU-Y-A3T), which is a combination of mutations that replace the amino acid at the position with glutamine (hereinafter referred to as 490Q) -4, BLU-Y-A3T-5), BLU-Y-A3T-1 Further confirmed that the improvement of the light emission amount can be seen, the present invention has been completed. That is, the present invention relates to the following.
(1) In firefly luciferase, the amino acid corresponding to position 344 of Heike firefly luciferase is mutated to alanine, the amino acid corresponding to position 287 is mutated to alanine, the amino acid corresponding to position 326 is mutated to serine, and corresponds to position 467 A firefly luciferase in which the amino acid to be mutated to isoleucine and the amino acid corresponding to position 75 is mutated to alanine.
(2) The firefly luciferase according to (1) above, wherein the amino acid corresponding to position 293 of Heike firefly luciferase is mutated to methionine.
(3) The firefly luciferase according to (1) above, wherein an amino acid corresponding to position 351 of Heike firefly luciferase is mutated to valine.
(4) The firefly luciferase according to (1) above, wherein an amino acid corresponding to position 172 of Heike firefly luciferase is mutated to glutamic acid and an amino acid corresponding to position 259 is mutated to isoleucine.
(5) The firefly luciferase according to (1) above, wherein an amino acid corresponding to position 359 of Heike firefly luciferase is mutated to valine and an amino acid corresponding to position 490 is mutated to glutamine.
(6) A firefly luciferase gene encoding the firefly luciferase according to (1) to (5) above.
(7) A recombinant DNA obtained by inserting the firefly luciferase gene described in (6) above into a vector DNA.
(8) A microorganism comprising the firefly luciferase gene described in (6) above or the recombinant DNA described in (7) above, cultivating a microorganism capable of producing firefly luciferase, and collecting firefly luciferase from the culture A method for producing firefly luciferase.

It is a graph which shows the relative comparison of the light-emission quantity in various firefly luciferases.

Hereinafter, the present invention will be described in detail.
(Firefly luciferase gene and its recombinant DNA)
As the firefly luciferase gene and its recombinant DNA of the present invention, those derived from any firefly can be used. For example, firefly luciferase derived from Heike firefly, Genji firefly, North American firefly, etc. can be used. Alternatively, chimeric genes prepared based on various firefly-derived luciferase genes may be used.

  Furthermore, the firefly luciferase gene of the present invention may contain a mutation other than the mutation of the present invention. The mutation may be artificially introduced with some specific effect, or may be randomly or non-artificially introduced. Mutations introduced with the intention of specific effects include, for example, addition and modification of sequences to enhance the expression level of the firefly luciferase gene, modification to improve the purification efficiency of firefly luciferase protein, Various mutations conferring practically favorable characteristics on luciferase can also be included. Examples of such known mutations include mutations that enhance luminescence persistence as described in JP 2000-197484 A, patents 2666561 or JP 2003-512071 A, and the like. Mutations that change the emission wavelength, mutations that increase the surfactant resistance as described in JP-A-11-239493, WO99 / 02697 pamphlet, JP10-512750A or JP2001-2001 Mutations that increase the substrate affinity as described in Japanese Patent No. 518799, or described in Japanese Patent No. 3048466, Japanese Patent Application Laid-Open No. 2000-197487, Japanese Patent Publication No. 9-510610 and Japanese Patent Publication No. 2003-518912 Such as mutations that increase stability.

  These genes and their recombinant DNA can be prepared according to known methods. For example, the Heike firefly luciferase gene and its recombinant DNA can be prepared by the method described in Japanese Patent Publication No. 7-112434, and the Genji firefly luciferase gene and its recombinant DNA are disclosed in JP-A-1-51086. It can be prepared by the method described in the publication, and further, the North American firefly luciferase gene and its recombinant DNA can be purchased from Promega.

(Gene variation and corresponding amino acid sequence variation in the present invention)
The firefly luciferase gene of the present invention is characterized by introducing a specific mutation into any of the above firefly luciferase genes. Specifically, one of the mutant genes of the present invention is the 344A mutation in which the amino acid at position 344 of luciferase (in the case of wild type, leucine) is mutated to alanine in the case of Heike firefly or Genji firefly, and the amino acid at position 287 The 287A mutation in which valine was mutated to alanine (in the case of the wild type), the 326S mutation in which amino acid at position 326 (which is glycine in the case of the wild type) was mutated to serine, the amino acid at position 467 (wild In the case of the type, it encodes a mutant firefly luciferase having a 467I mutation mutated to isoleucine and an amino acid at position 75 (valine in the case of the wild type) having a 75A mutation mutated to alanine. It is a mutant firefly luciferase gene.
Furthermore, in addition to the above-mentioned 5-fold mutation, a mutant firefly luciferase gene encoding a 293M mutant amino acid sequence in which the amino acid at position 293 (in the wild type, leucine) is mutated to methionine, or the amino acid at position 351 ( Mutated firefly luciferase gene encoding 351V mutant amino acid sequence mutated to valine in the case of wild type or amino acid at position 172 (lysine in the case of wild type) is mutated to glutamic acid Or a mutant firefly luciferase gene encoding a mutant amino acid sequence comprising a 259I mutation in which the amino acid at position 259 and the amino acid at position 259 (or threonine in the wild type) is mutated to isoleucine, or the amino acid at position 359 (wild type If aspartic acid is valine) Mutated 359V mutation and 490 of amino acids (in the case of wild-type, glutamic acid) is a mutant firefly luciferase gene encoding the variant amino acid sequence comprising a 490Q mutations mutated to glutamine.

  The amount of luminescence of firefly luciferase is significantly improved by having one or more amino acid mutations among 75A mutation, 351V mutation, 172E mutation, 259I mutation, 359V mutation and 490Q mutation in the amino acid sequence of Heike firefly luciferase or Genji firefly luciferase To do. In addition, when the amino acid sequence of the firefly luciferase of the present invention is combined with another mutation that reduces the amount of luminescence, such as the 344A mutation, in the amino acid sequence of firefly luciferase, the reduced amount of luminescence can be effectively recovered. In addition, when it is introduced in combination with another mutation, for example, the 287A mutation, the 326S mutation, and the 467I mutation, an excellent light emission amount improving effect can be obtained.

  By having 75A mutation, 293M mutation, 351V mutation, 172E mutation and 259I mutation, or 359V mutation and 490Q mutation in the amino acid sequence of Heike firefly luciferase or Genji firefly luciferase, the amount of luminescence of firefly luciferase is remarkably improved. The amino acid sequence of the firefly luciferase of the present invention preferably has at least one of the above substitutions in the amino acid sequence of firefly luciferase, more preferably two or more, and even more preferably three or more substitutions. What has is preferable. By introducing these multiple mutations, the amount of firefly luciferase emission can be increased stepwise. Specifically, in addition to the 75A mutation, combining one or more of the 293M mutation, the 351V mutation, the 172E mutation, the 259I mutation, or the 359V mutation and the 490Q mutation further increases the degree of improvement in the light emission amount of firefly luciferase.

  And when these mutations are combined with another mutation that reduces the light emission amount of firefly luciferase, for example, the 344A mutation when introduced, the reduced light emission amount can be effectively recovered. In particular, introducing a combination of 75A mutations into a firefly luciferase having a 344A mutation, or introducing a combination of one or more of 293M mutation, 351V mutation, 172E mutation and 259I mutation, or 359V mutation and 490Q mutation, Recovers the reduced luminescence of firefly luciferase. That is, the mutation of the present invention improves the amount of light emission of firefly luciferase compared to before the introduction of the mutation, and also exhibits excellent luminescence when introduced in combination with another mutation such as the 287A mutation, the 326S mutation, and the 467I mutation. The amount is improved.

  The light emission amount in the present invention means the light emission intensity when the firefly luciferase is reacted with firefly luciferin in the presence of ATP, divalent metal ions and oxygen unless otherwise specified. It can be determined that the greater the amount of luminescence, the stronger the firefly luciferase activity of the firefly luciferase. The amount of luminescence of the firefly luciferase can be evaluated using a relative luminescence intensity (RLU) obtained by using a desired luminescence measuring device, for example, a luminometer (LB9507 or LB96V, manufactured by Bertoled). The improvement in the amount of luminescence in the present invention refers to the case where the amount of luminescence when firefly luciferase is allowed to act on a predetermined condition shows an improvement of 1.2 times or more compared to that in which the mutation of the present invention is not introduced. Such a degree of improvement in the amount of luminescence in the present invention is remarkable in an attempt to introduce a mutation in order to improve the amount of luminescence of firefly luciferase, and is difficult to achieve easily. This is a useful improvement in practice.

(Number in the gene sequence and amino acid sequence of firefly luciferase)
In the present invention, the numbers indicating the positions of mutations in the gene sequence and amino acid sequence of firefly luciferase are based on the numbers in wild-type Heike firefly luciferase or Genji firefly luciferase. That is, when the present invention is applied to a luciferase other than wild-type Heike firefly luciferase, the mutation position in the gene sequence and amino acid sequence is changed with various firefly luciferases when the corresponding positions in wild-type Heike firefly luciferase or Genji firefly luciferase are replaced. The corresponding position. As specific examples, amino acid positions corresponding to positions 75, 293, 351, 172, 259, 359, and 490 of Heike firefly luciferase or Genji firefly luciferase are as follows: They are 73rd, 291st, 349th, 170th, 257th, 357th, and 488th.

  These correspondences can be determined, for example, by comparing the amino acid sequences of various luciferases with the amino acid sequences of Heikebotaru luciferase using, for example, software for analyzing homology of existing amino acids such as GENETYX-Mac (manufactured by Software Development). It can be easily known. In fact, common commonities in amino acid sequences and structural commonities have been found in Heike firefly luciferase, Genji firefly luciferase, and North American firefly luciferase, and similar mutations must be introduced at the corresponding positions. However, there are also known examples that have the same effect on the properties of firefly luciferase. Therefore, using the knowledge of the mutation of the present invention shown in Heike firefly luciferase, as a measure to obtain the same effect in Geni firefly luciferase or North American firefly luciferase, an attempt to introduce a similar mutation at the corresponding position It can be done easily.

(Introduction of mutation)
The above mutant firefly luciferase gene can be appropriately obtained by modifying the firefly luciferase gene by any known method. As a gene modification method, a method of introducing a mutation in a site-specific manner, a method of introducing a mutation at random, a method of causing a drug to act as a mutagen, an ultraviolet irradiation method, a protein engineering method, and the like can be widely used.

  Examples of the mutagen used in the above-described mutagenesis include hydroxylamine, N-methyl-N′-nitrosoguanidine (NTG), nitrous acid, sulfurous acid, hydrazine, formic acid, 5-bromouracil and the like. it can. The drug treatment is not particularly limited as long as it is possible to adopt suitable conditions depending on the type of drug used and the like, and a desired mutation can be induced in the firefly luciferase gene. For example, a desired mutation can be induced by treatment at 20 to 80 ° C. for 10 minutes or more, specifically 10 to 180 minutes at the above drug concentration of 0.5 to 12M.

  Ultraviolet irradiation can be performed using a known method such as a method described in Hyundai Kagaku, pp 24-30, June 1989.

  As a method of making full use of a protein engineering technique, a technique generally known as Site-specific Mutagenesis can be used. For example, Kramer method (Kramer, W. et al., Nucleic Acids Res, vol. 12, pp. 9441-9456 (1984): Kramer, W. et al., Methods Enzymol, vol. 154, pp. 350-367. (1987): Bauer, CE et al., Gene, vol. 37, pp. 73-81 (1985)), Eckstein method (Taylor, JW et al., Nucleic Acids Res, vol. 13). 87, 8764 (1985): Taylor, JW et al., Nucleic Acids Res, vol.13, pp. 8765-8785 (1985): Nakamaye, KL et al., Nucleic Aci. ds Res, vol. 14, pp. 9679-9698 (1986)), Kunkel method (Kunkel, TA, Proc. Natl. Acids Sci. USA, vol. 82, pp. 488-492). (1985): Kunkel, TA et al., Methods Enzymol, vol.154, pp.367-382 (1987).

  In addition, a technique generally known as Polymerase Chain Reaction can be used [Technique, 1, p. 11 (1989)].

  It is of course possible to directly synthesize a desired modified firefly luciferase gene by an organic synthesis method or an enzyme synthesis method in addition to the above gene modification method. The determination and confirmation of the base sequence in the desired firefly luciferase gene obtained by the above method can be carried out, for example, by the chemical modification method of Maxam-Gilbert [Maxam-Gilbert, Meth. Enzym. , Vol. 65, pp. 499-560 (1980)] or dideoxynucleotide chain termination method using M13 phage [Messing et al. Gene, vol. 19, pp. 269-276 (1982)].

(Preparation of vector and preparation of transformant)
The mutant firefly luciferase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of prokaryotic cells or eukaryotic cells according to a conventional method, Vectors can be used for transformation or transduction.
Although any host can be used, for example, a microorganism is preferable. Specifically, microorganisms belonging to the genus Escherichia can be used. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli K-12, JM109, DH5α, HB101, BL21 and the like.

  A strain having the ability to produce mutant firefly luciferase can be obtained by screening a strain having the ability to produce firefly luciferase having a desired mutation from the obtained transformant or the transduced host. In order to obtain a purified novel recombinant DNA from the strain thus obtained, Guerry's method [J. Bacteriology, 116, p. 1604 (1973)], Clewell's method [J. Bacteriology, 110, p. 667 (1972)] and the like can be used.

  In order to obtain DNA containing the mutant firefly luciferase gene from the obtained recombinant DNA, various restriction enzymes can be added to the plasmid DNA at a reaction temperature of 30 to 40 ° C. A method may be used in which the reaction-terminated solution is subjected to agarose gel electrophoresis after acting for 24 hours (see Molecular Cloning, 150, Cold Spring Harbor Laboratory (1982)).

(Production of mutant firefly luciferase)
In order to produce mutant firefly luciferase using the strain having the ability to produce mutant firefly luciferase of the present invention obtained as described above, the strain having ability to produce mutant firefly luciferase is cultured by various known methods. To do. The culture may be a solid culture method, but is preferably cultured using a liquid culture method.

Examples of the medium for culturing the strain include yeast extract, tryptone, peptone, meat extract, corn steep liquor, or one or more nitrogen sources such as soybean or wheat bran leachate, sodium chloride, potassium monophosphate One or more inorganic salts such as dibasic potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate were added, and saccharide raw materials, vitamins, etc. were added as necessary Things are used.
The initial pH of the medium is suitably adjusted to pH 7-9. Cultivation is preferably performed at 30 to 40 ° C., preferably around 37 ° C. for 4 to 24 hours, preferably 6 to 8 hours, by aeration and agitation culture, shaking culture, stationary culture, or the like. In order to collect firefly luciferase with improved stability from the culture after completion of the culture, an ordinary known enzyme collecting means may be used.

  Specifically, the cells are subjected to ultrasonic disruption treatment, grinding treatment, etc. by a conventional method, or a firefly luciferase with improved luminescence is extracted using a lytic enzyme such as lysozyme, or organic such as toluene. In the presence of a solvent, it can be shaken or allowed to stand for self-digestion to discharge firefly luciferase out of the cells. The obtained extract or autolysate is subjected to filtration, centrifugation, etc. to remove solid components. If necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate, etc., and then ammonium sulfate, alcohol, Acetone or the like is added for fractionation to obtain a crude enzyme.

  For example, gel filtration using Sephadex, Ultrogel or biogel; adsorption elution using ion exchanger; electrophoresis using polyacrylamide gel; adsorption elution using hydroxyapatite; sucrose density gradient Enzyme standards with a further increased degree of purification by appropriately selecting known techniques such as sedimentation methods such as centrifugation; affinity chromatography; fractionation using molecular sieve membranes or hollow fiber membranes, etc. Product.

  Hereinafter, the present invention will be described more specifically with reference to examples.

1. Preparation of plasmid pET16b-BLU-Y A full length primer (SEQ ID NO: 1) for amplifying a biotinylated luciferase structural gene was synthesized. The plasmid pHLf248 described in Japanese Patent No. 3466765 (E. coli JM101 [pHLf248] including this plasmid is deposited as FERM BP-5081 at the National Institute of Advanced Industrial Science and Technology (AIST) Patent Biological Depositary), as a template. The DNA fragment was amplified by PCR using a full length primer and a commercially available M13-M4 primer (manufactured by Takara Bio Inc.). The obtained DNA fragment was digested with NdeI and HindIII and then subjected to agarose gel electrophoresis to purify the DNA fragment from a 1.9 kb band.

Next, the above DNA fragment was inserted into NdeI and HindIII digested plasmid vector pET16b (manufactured by Novagen) by a conventional method to prepare plasmid pET16b-BLU-Y.
Further, the prepared plasmid pET16b-BLU-Y was introduced into E. coli strain JM109 and transformed. After purifying the plasmid from the obtained transformant, the DNA sequence was confirmed. The amino acid sequence of firefly luciferase (BLU-Y) deduced from the firefly luciferase gene contained in this DNA sequence was identical to that described in Japanese Patent No. 3466765.

2. Preparation of plasmid containing mutant firefly luciferase gene)
In the amino acid sequence of Heike firefly luciferase, leucine, which is the 344th amino acid residue, is mutated to alanine (mutation 344A), alanine, which is the 287th amino acid residue, is mutated to valine (mutation 287V), and the 326th amino acid residue. A plasmid (pHLfA3T) containing a gene encoding luciferase in which glycine was mutated to serine (G326S mutation) and phenylalanine 467th amino acid residue was mutated to isoleucine (F467I) was prepared as follows.

  First, PCR primers (pHLf344A F-344A (SEQ ID NO: 2), pHLf344A R-344A (SEQ ID NO: 3)) designed to convert leucine, the 344th amino acid residue, into alanine were synthesized. Using these primers, PCR amplification was carried out using pET16b-BLU-Y as a template (reaction procedure and conditions were in accordance with standard methods). The amplified PCR reaction solution was digested with DpnI, and the end of the PCR product was phosphorylated by kinase treatment. Subsequently, the PCR product reaction product after DpnI and kinase treatment was self-ligated using Ligation Convenience Kit (manufactured by Nippon Gene) to obtain pHLf344A. Subsequently, PCR primers (pHLf287A F-287A (SEQ ID NO: 4), pHLf287A R-287A (SEQ ID NO: 5)) designed to convert valine at the 287th amino acid residue into alanine were synthesized. PCR amplification using pHLf344A as a template was performed using these primers, and pHLf344A / 287A was obtained according to the method described above. Subsequently, primers (pHLf326S F-326S (SEQ ID NO: 6), pHLf326S R-326S (SEQ ID NO: 7)) designed to convert glycine, which is the 326th amino acid residue, into serine, and the 467th amino acid. PCR amplification was performed using primers (pHLf467I F-467I (SEQ ID NO: 8), pHLf467I R-467I (SEQ ID NO: 9)) designed to convert phenylalanine to isoleucine, and pHLf-BLU-Y according to the above method. -A3T was obtained.

D. M.M. According to Morrison's method (Methods in Enzymology, 68, p. 326-331, 1979), E. coli JM109 strain was transformed with this pHLf-BLU-Y-A3T, and a plasmid was obtained from the resulting transformant JM109 strain. After purification, the DNA sequence encoding the mutant luciferase in pHLf-BLU-Y-A3T was confirmed (SEQ ID NO: 10). In the amino acid sequence deduced from this DNA sequence, leucine, the 344th amino acid residue in the amino acid sequence corresponding to the luciferase gene contained in pET16b-BLU-Y obtained in Example 1, is converted to alanine. The 287th amino acid residue valine was substituted with alanine, the 326th amino acid residue glycine was substituted with serine, and the 467th amino acid residue phenylalanine was substituted with isoleucine. .
All of the plasmids containing a gene encoding a luciferase obtained by artificially combining the mutations found in the present invention, or a luciferase in which the mutation found in the present invention is substituted with another amino acid, follow the above procedure. Made.

3. Acquisition of luminescence-enhancing mutant firefly luciferase)
Using the recombinant plasmid pHLf-BLU-Y-A3T described in Example 2 as a template, error-prone PCR was performed using primers (SEQ ID NOs: 11 and 12) designed upstream and downstream of the mutant luciferase gene region. Specifically, using these primers at a final concentration of 0.2 μM and using Ex-Taq (manufactured by Takara Bio Inc.) under a manganese ion concentration of 0.1 mM and a magnesium ion concentration of 6.5 mM, pHLf-BLU A firefly luciferase gene fragment into which various mutations were introduced was obtained by performing a PCR amplification reaction on the -Y-A3T gene region. These were then treated with restriction enzymes with NdeI and HindIII, separated by agarose gel electrophoresis, and DNA fragments were recovered from the gel after electrophoresis using RECO-CHIP (Takara Bio Inc.). The obtained DNA fragment was ligated to pHLf-BLU-Y-A3T vector obtained by treating pHLf-BLU-Y-A3T with NdeI and HindIII using Ligation Convenience Kit (Nippon Gene). After the ligation, according to the method described above, E. coli BL21 (DE3) (manufactured by Invitrogen) is transformed with the recombinant plasmid DNA into which the mutation has been introduced to produce a mutant library. did. This transformed strain was treated with LB-amp agar medium [1% (w / v) bactotryptone, 0.5% (w / v) yeast extract, 10.5% (w / v) NaCl, and 50 μg / ml ampicillin. And 1.4% (w / v) agar] and plated at 37 ° C.

After 12 hours, each colony that appeared was treated with LB-amp liquid medium [1% (w / v) bactotryptone, 0.5% (w / v) yeast extract, 10.5% (w / v) NaCl. And 50 μg / ml ampicillin] and statically cultured at 37 ° C. in a 96-well plate. After culturing for 12 to 18 hours, the cells are lysed using a lysing agent (Bag Buster, Novagen), and then an activity measurement reagent [50 mM Tricine-NaOH, 4 mM ATP, 2 mM Luciferin, 10 mM MgSO ₄ , pH 7.8. The activity of firefly luciferase contained in each strain was confirmed using a luminometer (LB96V, manufactured by Bertoled). In the above measurement, firefly luciferase whose firefly luciferase activity, that is, the amount of luminescence was increased by 1.2 times or more as compared with the parent strain before the introduction of the mutation was selected as a candidate for the luminescence enhancement mutant.

4). Confirmation of mutation position The strain selected in Example 3 was cultured in 2 ml of LB-IPTG-amp medium. After culturing for 18 to 24 hours, the cells were collected by centrifugation, and the cells were collected with 50 mM potassium phosphate buffer, 0.2% (w / v) BSA (manufactured by Wako Pure Chemical Industries, Ltd.), pH 7.5. Was suspended and subjected to ultrasonic crushing to obtain a crude enzyme solution. The activity of the luciferase crude enzyme was measured for the above crude enzyme solution using an activity measurement reagent [50 mM Tricine-NaOH, 4 mM ATP, 2 mM Luciferin, 10 mM MgSO ₄ , pH 7.8]. The luciferase activity was evaluated by the amount of luminescence obtained by integration for 1 second using a luminometer (Beltoled, LB96V). A candidate strain having a greater activity than the parent strain was used as a luminescence enhancement mutant, and the sequence of the firefly luciferase gene encoded by the plasmid in the stability enhancement mutant was determined using CEQ2000 DNA Sequencing System (manufactured by Beckman Coulter).

As a result, the corresponding gene sequences in the five candidate strains were each replaced with amino acid sequence (SEQ ID NO: 13) in which valine at position 75 was replaced with alanine, or / and leucine at position 293 was replaced with methionine. Amino acid sequence (SEQ ID NOs: 14 and 15), amino acid sequence (SEQ ID NO: 16) in which isoleucine at position 351 is replaced with valine, amino acid in which lysine at position 172 is replaced with glutamic acid, and threonine at position 259 is replaced with isoleucine It encodes a mutant firefly luciferase consisting of the sequence (SEQ ID NO: 17) or an amino acid sequence (SEQ ID NO: 18) in which the aspartic acid at position 359 is replaced with valine and the glutamic acid at position 490 is replaced with glutamine. confirmed. Codes the amino acid sequence of firefly luciferase in which leucine at position 344 is alanine, valine at position 287 is alanine, glycine at position 326 is serine, phenylalanine at position 467 is replaced with isoleucine, and valine at position 75 is replaced with alanine The gene sequence is shown in SEQ ID NO: 19.
Furthermore, using the information on the mutation point in the obtained mutant strain, in accordance with the method described in the previous example, a new mutant strain having a mutation combining a plurality of the above mutations, or replacing the mutation with another amino acid Mutant strains were prepared and various mutant firefly luciferases were obtained.

5. Evaluation of luminescence amount of mutant firefly luciferase According to the method described in Example 4, the luciferase activity of mutant firefly luciferases having various amino acid sequences obtained was measured to evaluate the luminescence amount. The result is shown in FIG.

  In the product not containing the mutation of the present invention (BLU-Y A3T), the amount of luminescence of the crude enzyme was 85,0670 RLU, while that having the 75A mutation (BLU-Y-A3T-1, B-A3T in the figure) -1), which is a significant improvement (1.3 times) to 1,101,574 RLU. Thus, it was found that the 75A mutation is highly effective for improving the amount of light emission of firefly luciferase. Those having 75A mutation and 293M mutation (BLU-Y-A3T-2, described as B-A3T-2 in the figure) have 1,334,766RLU, 75A mutation and 351V mutation (BLU-Y-A3T). -3, described as B-A3T-3 in the figure) having 1,581,320 RLU, 75A mutation, 172E mutation and 259I mutation (BLU-Y-A3T-4, described as B-A3T-4 in the figure) 2,135,263 RLU, and those having 75A mutation, 359V mutation, and 490N mutation (BLU-Y-A3T-5, indicated as B-A3T-5 in the figure), 1,327,261 RLU, and mutations of the present invention The amount of luminescence was remarkably improved (1.5 to 2.5 times) as compared with the case of not containing. Thus, it was found that the mutation of the present invention is highly effective for improving the amount of firefly luciferase.

6). Luminescence persistence of mutant firefly luciferase The luminescence persistence of each mutant tested in Example 5 was confirmed using a luminometer (LB96V, manufactured by Bertoled Co., Ltd. [Reaction reagent: 50 mM Tricine-NaOH, 0.8 mM ATP, 0 5 mM Luciferin, 10 mM MgSO ₄ , 0.2% BSA, 2% Sucrose, 1 mM EDTA]. Luminescence persistence was measured from 1.2 seconds to 60 seconds after the start of measurement, and compared by the ratio of the measured value after 60 seconds to the measured value after 1.2 seconds (luminance rate persistence). That is, the lower the ratio of the measured value after 60 seconds to the measured value after 1.2 seconds, the greater the degree of light emission attenuation and the worse the light emission persistence.

  As a result, in the firefly luciferase (BLU-Y) not containing the L344A mutation, the luminescence persistence was 27.8%, and after 60 seconds, the luminescence decreased to less than one third, whereas the luminescence persistence mutation In all mutant firefly luciferases containing the L344A mutation, the luminescence persistence was 100-150%. That is, all of the mutants containing the stability mutation of the present invention and containing the L344A mutation exhibit the effect of improving the amount of luminescence while maintaining the luminescence persistence that is the effect of the L344A mutation. found.

7). Thermal stability of mutant firefly luciferase)
A crude enzyme solution of each mutant tested in Example 5 was added with 1 to 10 μl of 0.3 M Tricine-NaOH, 0.2% BSA, 5% Glycerol, pH 7.8, at a reaction temperature of 47 ° C. For 90 minutes. Then, the activity of the luciferase crude enzyme before and after the heat treatment was measured using an activity measurement reagent [50 mM Tricine-NaOH, 4 mM ATP, 2 mM Luciferin, 10 mM MgSO ₄ , pH 7.8]. The luciferase activity was evaluated by the amount of luminescence obtained by integrating for 1 second using a luminometer (Berthold, LB96V), and the ratio of the amount of luminescence after heat treatment to the amount of luminescence before heat treatment was expressed as “activity remaining. Calculated as "rate".
As a result, BLU-Y A3T had a residual activity rate of 34.4%, whereas BLU-A3T-1 was 37.3%, B-A3T-2 was 38.3%, B-A3T- 3 was 50.7%, BLU-A3T-4 was 50.6%, and BLU-A3T-5 was 52.7%. That is, even if a luminescence improvement mutation of 75A mutation, or a luminescence improvement mutation combining one or more of 293M mutation, 351V mutation, 172E mutation and 259I mutation, or 359V mutation and 490Q mutation in addition to the 75A mutation, It has been found that the high thermal stability, which is the effect of the 344A mutation, the 287A mutation, the 326S mutation, and the 467I mutation, is well maintained, but rather tends to be further improved.

  As can be seen from these results, the method of the present invention can provide firefly luciferase with excellent light emission. In addition, firefly luciferase having properties such as luminescence persistence and stability, and a high light emission amount can be provided. According to the present invention, firefly luciferase having an excellent light emission amount is provided and can be advantageously used in a kit such as a high-sensitivity luminescence measurement system or a trace ATP measurement system, and also a luminescence persistence mutation, a stability improvement mutation, etc. By combining with other useful mutations, further widespread use is expected.

Claims (8)

In firefly luciferase, the amino acid corresponding to position 344 of Heike firefly luciferase is mutated to alanine, the amino acid corresponding to position 287 is mutated to alanine, the amino acid corresponding to position 326 is mutated to serine, and the amino acid corresponding to position 467 is Firefly luciferase mutated to isoleucine and an amino acid corresponding to position 75 is mutated to alanine. The firefly luciferase according to claim 1, wherein an amino acid corresponding to position 293 of Heike firefly luciferase is mutated to methionine. The firefly luciferase according to claim 1, wherein an amino acid corresponding to position 351 of Heike firefly luciferase is mutated to valine. The firefly luciferase according to claim 1, wherein the amino acid corresponding to position 172 of Heike firefly luciferase is mutated to glutamic acid and the amino acid corresponding to position 259 is mutated to isoleucine. The firefly luciferase according to claim 1, wherein the amino acid corresponding to position 359 of Heike firefly luciferase is mutated to valine and the amino acid corresponding to position 490 is mutated to glutamine. The firefly luciferase gene which codes the firefly luciferase of Claims 1-5. Recombinant DNA, wherein the firefly luciferase gene according to claim 6 is inserted into vector DNA. A firefly luciferase comprising the firefly luciferase gene according to claim 6 or the recombinant DNA according to claim 7, cultivating a microorganism having firefly luciferase-producing ability, and collecting firefly luciferase from the culture. Manufacturing method.

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