WO2007144990A1 - 変異型ルシフェラーゼ - Google Patents
変異型ルシフェラーゼ Download PDFInfo
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- WO2007144990A1 WO2007144990A1 PCT/JP2007/051279 JP2007051279W WO2007144990A1 WO 2007144990 A1 WO2007144990 A1 WO 2007144990A1 JP 2007051279 W JP2007051279 W JP 2007051279W WO 2007144990 A1 WO2007144990 A1 WO 2007144990A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Definitions
- the present invention relates to a mutant luciferase having a luciferase activity with a changed emission spectrum, for example.
- Reporter assembly is one of means for quantifying the transcriptional activity of a transcriptional regulatory sequence.
- reporter gene a gene encoding a reporter protein (hereinafter referred to as “reporter gene”) is linked under the control of the transcriptional control sequence (promoter, enhancer, etc.) to be examined, introduced into a host cell, and expressed.
- transcriptional control sequence promoter, enhancer, etc.
- Reporter assembly can be performed using various proteins as reporter proteins.
- the fluorescent protein is a reporter protein
- the relative amount of the reporter protein can be quantified by irradiating the expressed fluorescent protein with excitation light and measuring the intensity of the generated fluorescence (fluorescence). Called the law).
- reporter assembly can be performed using an enzyme such as j8-galactosidase or alkaline phosphatase as a reporter protein.
- the enzyme is a reporter protein
- the relative amount of the reporter protein can be quantified by colorimetry by using a substrate that is decomposed by the action of the enzyme and generates a color substance (called colorimetric method).
- colorimetric method a substrate that is decomposed by the action of the enzyme and generates a color substance
- the relative amount of reporter protein can be quantified by measuring the amount of luminescence (referred to as luminescence method).
- the light emitting method has the following excellent features. First, since no excitation light is required unlike the fluorescence method, a high signal / noise ratio with a small knock ground can be obtained. In addition, it has high sensitivity and a wide dynamic range. Furthermore, the quantitative property is excellent. [0006]
- An enzyme reaction system generally used in the luminescence method includes a luciferase / luciferin reaction system.
- luciferases derived from various organisms including fireflies and renilla.
- Each luciferase is limited to some extent in the type of luciferin recognized as a substrate.
- a technique commonly referred to as Dual Reporter Atsease a sample containing a mixture of firefly-derived luciferase (hereinafter referred to as ⁇ firefly luciferase ''! And rumilla mushroom-derived luciferase (hereinafter referred to as ⁇ Rumilla luciferase '').
- Firefly luciferase and firefly luciferin (hereinafter referred to as ⁇ firefly luciferin '') and luciferin derived from renilla (hereinafter referred to as ⁇ renilla luciferin '') (coelenterazine) are added to the solution in order. Measure each activity of Renilla luciferase separately.
- the sea fireflies include species such as Vargula hilgendorffi and Cypridina noctiluca.
- luciferase is released outside the body (that is, in seawater), and luciferin reacts with oxygen in seawater due to the catalytic action of luciferase to emit light.
- VLuc Valgra Hilgendolphy
- CLuc sipridina 'noctil force
- luciferase gene when used as a reporter gene, it is possible to measure the transcriptional activity of a transcription control sequence such as a promoter without disrupting the cells (international Published 2006/132350 Pan Fret).
- the secretory luciferase can be used as a test solution as it is, and is suitable for constructing a so-called high-throughput reporter assay system that processes a large number of samples.
- non-secretory luciferase it is essential to collect cells by centrifugation and crush cells (or to increase cell permeability) by sonication, surfactant treatment, organic solvent treatment, etc.
- CLuc is secreted into the culture medium 320 times when expressed in NIH3T3 cells and 410 times when expressed in HeLaS 3 cells compared to VLuc. (Non-patent document 2). Therefore, compared to VLuc, CLuc is suitable for use in a high-sensitivity, high-throughput reporter assay system using cultured cells as a host!
- the luminescence mechanism of the luciferase / luciferin reaction system is generally considered as follows. First, luciferin is oxidized to oxyluciferin in an excited state by the catalytic action of luciferase. Next, the excited state oxyluciferin immediately returns to the ground state, but releases (emits) energy as light in the process. The amount of luminescence per unit time at this time is considered to be proportional to the amount of luciferase present in the system, and the relative amount of luciferase can be quantified by luminescence.
- multi-reporter assembly there are at least the following two methods for simultaneously performing two or more promoters with one activity of reporter assembly using a luciferase gene as a reporter gene (referred to as multi-reporter assembly).
- a method using luciferin of a plurality of different chemical species and a luciferase having substrate specificity for each luciferin there is a method using luciferin of a plurality of different chemical species and a luciferase having substrate specificity for each luciferin.
- the conditions (reaction solution composition, hydrogen ion concentration, etc.) suitable for each luciferase / luciferin reaction system reaction are also different.
- this method has a problem when the measurement operation becomes complicated.
- luciferin of the same chemical species As a second method, there is a method using luciferin of the same chemical species as a substrate.
- a plurality of types of luciferases using luciferin of the same chemical species as substrates are used as reporter proteins.
- the amino acid sequences of these luciferases are partially different, and each luciferase is characterized by producing different emission spectra. The intensity of luminescence derived from each luciferase needs to be discriminated and quantified by the difference in spectrum.
- the multi-reporter assembly using the second method has an advantage of being simple because it can use only one kind of substrate and can perform the luminescence reaction and the measurement at a time.
- a multi-reporter assembly using the principle of the second method is derived from a luminescent beetle.
- a luminescent beetle There is one using a luciferase gene and its mutant gene (Non-patent Document 4).
- the luminescent beetle-derived luciferase is non-secretory. Therefore, it is not suitable for high throughput due to the reasons described above.
- BRET Bioluminescence resonance energy transfer
- f column a method for detecting structural changes in proteins at the biochemical level or the cell level
- a light emitter and a fluorophore form a pair.
- bioluminescence such as luciferase / lucifrin is used as the illuminant.
- the fluorophore for example, a fluorescent chemical substance or a fluorescent protein such as green fluorescent protein (GFP) is used.
- GFP green fluorescent protein
- each phosphor has its own excitation spectrum, and the excitation efficiency depends on the emission spectrum of the phosphor and the excitation spectrum of the phosphor.
- a phosphor that emits light having a wavelength that efficiently excites the phosphor is most preferable.
- the luciferase used uses light of a wavelength that efficiently excites the luminescent material with reference to the excitation spectrum of the fluorescent material used as a pair. What emits is preferred. Therefore, the presence of mutant luciferases with different wavelengths of emitted light makes it possible to form appropriate BRET pairs for various fluorophores.
- Patent Document 1 Japanese Patent Laid-Open No. 3-30678
- Patent Document 2 JP 2004-333457 A
- Non-Special Reference 1 Thompson, EM, Nagata S., Tsuji FI, “Proceedings of the National Academy of Sciences of the United States of America J, 1989, 86th, p.6567—65
- Non-Patent Document 2 Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., Ohmiya, Y., [Bioscience and Biotechnology and BiochemistryJ, 2004, Brother 8 ⁇ , p.565-570
- Non-Patent Document 3 Viviani, V "Uchida, A” Suenaga, N "Ryuluku, M., Ohmiya, Y", Bioc hemistry and Biophysics Research Communication J, 2001, No. 280, p.1286- 1291
- Non-Patent Document 4 Yoshihiro Nakajima, Katsuhiro Omiya, “Biotechnology Journal”, 2006, Vol. 6, No. 2, p.230
- Non-Patent Document 5 Otsuji, T “Okuda-Ashitaka, E” Kojima, S “Akiyama, H” Ito, S., Ohmiya, Y., "Analytical Biochemistry J, 2004, No. 329, p.230-237
- an object of the present invention is to provide a mutant luciferase having a luciferase activity with a changed emission spectrum.
- wild-type luciferase is obtained by substituting specific amino acid residues in luciferase (CLuc) derived from Cypridina noctiluca.
- CLuc luciferase derived from Cypridina noctiluca.
- the inventors have found that a mutant luciferase having a luciferase activity that imparts a different emission spectrum can be obtained, and has completed the present invention.
- the present invention includes the following.
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 375th amino acid, and the light emission is 457 nm or more.
- a mutant luciferase comprising any one of the following (a) to (1):
- amino acid sequence of (a) above consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the 178th amino acid, and luminescence of 449 nm or less Protein with spectral peak luciferase activity
- a protein comprising an amino acid sequence in which the first to 18th amino acids are deleted and the 178th methionine is replaced with another amino acid in the amino acid sequence shown in SEQ ID NO: 2.
- amino acid sequence of (c) above consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added at a position other than the 178th amino acid, and luminescence of 449 nm or less Protein with spectral peak luciferase activity
- a mutant luciferase consisting of any one of the following (a) to (). [0037] (a) a protein comprising an amino acid sequence in which the 167th threonine is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 2
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 167th amino acid, and the light emission is 458 nm or more.
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 404th amino acid, and the light emission is 458 nm or more.
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 404th amino acid, and the light emission is 458 nm or more.
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 406th amino acid, and the light emission is 460 nm or more.
- amino acid sequence of (c) above consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the above amino acids, and has an emission spectrum peak of 466 nm or more.
- a mutant luciferase comprising the following (a) to (no! /, Shear force: one protein.
- amino acid sequence of (c) above consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the above amino acids, and has an emission spectrum peak of 435 nm or less.
- (33) comprising a step of contacting the culture or culture supernatant of the transformant according to (32) with luciferin or a derivative thereof, and a step of measuring the luminescence intensity of an emission spectrum based on each luciferase activity.
- a method for evaluating the transcriptional activity of a promoter comprising evaluating the transcriptional activity of the promoter.
- a mutant luciferase having a luciferase activity that imparts an emission spectrum different from that of a wild-type luciferase is provided.
- the mutant luciferase according to the present invention when used, a simple and highly sensitive multi-reporter assembly system can be provided.
- the mutant luciferase according to the present invention is an excellent energy donor.
- FIG. 1 shows relative luminescence intensity versus wavelength for each luciferase.
- the first mutant luciferase according to the present invention comprises any one of the following (a) to ( (Hereinafter referred to as “first mutant luciferase”).
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 375th amino acid, and the light emission is 457 nm or more. Protein with spectral peak luciferase activity.
- a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is luciferase (CLuc) derived from Cypridina noctyl force.
- the base sequence shown in SEQ ID NO: 1 is a gene (cDNA) encoding CLuc.
- the mutant luciferase described in (a) above in the first mutant luciferase is a protein comprising an amino acid sequence in which the 375th lysine is substituted with another amino acid in the amino acid sequence of CLuc.
- the protein exhibits luciferase activity like CLuc.
- the protein emits light during luciferin oxidation due to luciferase activity, and the emission spectrum peak due to CLuc is 454 nm, whereas it is 457 nm or more, particularly 457 ⁇ ! An emission spectrum peak of ⁇ 490 nm (for example, 457 ⁇ ! To 463 nm).
- the other amino acid any amino acid other than lysine may be used.
- the mutant luciferase described in (b) above in the first mutant luciferase is the mutant luciferase described in (a). 1-10 (preferably 1-5, particularly preferably 1-3) amino acid sequence having an amino acid deleted, substituted or added, and having a luminescence spectrum peak of 457 nm or more. It is what you have. Positions other than the 375th amino acid Examples of the position include the 167th threonine, the 403th glutamine, the 404th asparagine, the 405th threonine, the 406th serine, and the 407th isoleucine.
- the mutant luciferase described in (c) above has the amino acid sequence ability of the mutant luciferase described in (a) CLuc secretion signal peptide (of the amino acid sequence represented by SEQ ID NO: 2). It is a mature protein excluding the first to 18th amino acid sequences.
- secreted proteins including CLuc are synthesized in the form of a precursor having a secretory signal peptide at the N-terminus. This precursor is cleaved by a signal peptidase during the transmembrane process to become a mature protein.
- the mature protein means a protein secreted outside the cell membrane or outside the cell wall.
- the second mutant luciferase according to the present invention is any one of the following proteins (a) to (hereinafter referred to as “second mutant luciferase”).
- amino acid sequence of (a) above consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the 178th amino acid, and luminescence of 449 nm or less A protein having spectral peak luciferase activity;
- amino acid sequence of (c) above consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added at a position other than the 178th amino acid, and luminescence of 449 nm or less Protein with spectral peak luciferase activity.
- the mutant luciferase described in (a) above is CLuc
- the 178th methionine is a protein consisting of an amino acid sequence in which another amino acid is substituted.
- the protein exhibits luciferase activity like CLuc.
- the protein emits light during luciferin oxidation by luciferase activity!
- the emission spectrum peak by CLuc is 454 ⁇ m, whereas it is 449 nm or less, particularly 420 nm to 449 nm ( For example, the emission peak is 447 ⁇ ! ⁇ 449nm).
- the other amino acid may be any amino acid other than methionine, but lysine is particularly desirable.
- the mutant luciferase described in (b) above in the second mutant luciferase is the mutant luciferase described in (a), and in the mutant luciferase described in (a), one or more (for example, at a position other than the 178th amino acid) (1 to 10, preferably 1 to 5, particularly preferably 1 to 3) amino acid sequence having an amino acid deleted, substituted or added, and having a luminescence spectrum peak of 449 nm or less of luciferase activity It is what you have.
- the position other than the 178th amino acid include the 197th leucine.
- the mutant luciferase described in (c) above is a mature protein from which the secretory signal peptide of amino acid sequence CLuc of the mutant luciferase described in (a) is excluded.
- mutant luciferase described in (d) above in the second mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- the third mutant luciferase according to the present invention is one of the following proteins (a) to (hereinafter referred to as “third mutant luciferase”).
- amino acid sequence shown in SEQ ID NO: 2 the amino acid sequence consists of an amino acid sequence in which the 1st to 18th amino acids are deleted and the 167th threonine is replaced with another amino acid. Tannotype;
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 167th amino acid, and the light emission is 458 nm or more. Protein with spectral peak luciferase activity.
- the mutant luciferase described in (a) above in the third mutant luciferase is a protein comprising an amino acid sequence in which the 167th threonine is substituted with another amino acid in the amino acid sequence of CLuc.
- the protein exhibits luciferase activity like CLuc. However, by this amino acid substitution, the protein emits light upon luciferin oxidation by luciferase activity!
- the emission spectrum peak by CLuc is 454 ⁇ m, whereas it is 458 nm or more, particularly 458 nm to 490 nm ( For example, the emission spectrum peak is 458 ⁇ ! ⁇ 460nm).
- the other amino acid may be any amino acid other than threonine, and examples thereof include isoleucine, leucine, and lysine.
- the mutant luciferase described in (b) above in the third mutant luciferase is the mutant luciferase described in (a), and in the mutant luciferase described in (a), one or more (for example, at a position other than the 167th amino acid) 1-10 (preferably 1-5, particularly preferably 1-3) amino acid sequence having a deleted, substituted or added amino acid, and having a luminescence spectrum peak of 458 nm or more. It is what you have.
- positions other than the 167th amino acid include, for example, the 375th lysine, the 403th glutamine, the 404th arsenolagin, the 405th threonine, the 406th serine, the 407th Of isoleucine.
- the mutant luciferase described in (c) above in the third mutant luciferase is a mature protein from which the secretory signal peptide of the amino acid sequence ability CLuc of the mutant luciferase described in (a) is excluded.
- the fourth mutant luciferase according to the present invention is one of the following (a) to (1): (Hereinafter referred to as “fourth mutant luciferase”).
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 404th amino acid, and the light emission is 458 nm or more.
- a protein having spectral peak luciferase activity
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 404th amino acid, and the light emission is 458 nm or more. Protein with spectral peak luciferase activity.
- the mutant luciferase described in (a) above in the fourth mutant luciferase is a protein comprising an amino acid sequence in which the 404th asparagine is substituted with another amino acid in the CLuc amino acid sequence.
- the protein exhibits luciferase activity like CLuc.
- this amino acid substitution causes the protein to emit light during luciferase oxidation due to luciferase activity, whereas the emission spectrum peak due to CLuc is 454 nm, whereas 458 nm or more, particularly 458 nm to 490 nm (for example, Emission spectrum peak from 458nm to 460nm).
- the other amino acid may be any amino acid other than asparagine, and examples thereof include glycine, alanine, serine, and threonine, and glycine or serine is particularly desirable.
- one or more (for example, at a position other than the 404th amino acid) 1-10 (preferably 1-5, particularly preferably 1-3) amino acid sequence having a deleted, substituted or added amino acid, and having a luminescence spectrum peak of 458 nm or more. It is what you have.
- positions other than the 404th amino acid include the 38th lysine, the 45th serine, the 75th parin, the 79th arginine, the 87th arginine, and the 112th aspartic acid. No.
- 126 Lysine of eye 167th threonine, 170th glutamic acid, 191st mouth isine, 223th methionine, 235th glutamine, 258th parin, 276th isoleucine 280th tyrosine, 291st methionine, 313th threonine, 372rd arginine, 375th lysine, 403th glutamine, 405th threonine, 406th Serine, 407th isoleucine, and 479th glutamic acid.
- the mutant luciferase described in (c) above is a mature protein from which the secretory signal peptide of the amino acid sequence ability CLuc of the mutant luciferase described in (a) is excluded.
- mutant luciferase described in (d) above in the fourth mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- the fifth mutant luciferase according to the present invention is any one of the following proteins (a) to (hereinafter referred to as “fifth mutant luciferase”).
- the mutant luciferase described in (a) above in the fifth mutant luciferase is a protein comprising an amino acid sequence in which the 405th threonine is substituted with another amino acid in the CLuc amino acid sequence.
- the protein has a luciferase activity similar to CLuc. Showing gender. However, due to this amino acid substitution, the protein emits light upon luciferin oxidation by luciferase activity!
- the emission spectrum peak by CLuc is 454 ⁇ m, whereas 457 nm or more, especially 457 ⁇ ! The emission spectrum peak is ⁇ 490nm (for example, 457 ⁇ ! ⁇ 460nm).
- the other amino acid may be any amino acid other than threonine, and examples thereof include isoleucine, methionine, and leucine. In particular, isoleucine or methionine is desired.
- the mutant luciferase described in (b) above in the fifth mutant luciferase is the mutant luciferase described in (a), and in the mutant luciferase described in (a), one or more (for example, at a position other than the 405th amino acid) 1-10 (preferably 1-5, particularly preferably 1-3) amino acid sequence having an amino acid deleted, substituted or added, and having a luminescence spectrum peak of 457 nm or more. It is what you have.
- positions other than the 405th amino acid include the 38th lysine, the 45th serine, the 75th parin, the 79th arginine, the 87th arginine, and the 112th aspartic acid.
- 126th lysine, 167th threonine, 170th glutamic acid, 191st mouth isine, 223th methionine, 235th glutamine, 258th parin, 276th No. isoleucine, No. 280 tyrosine, No. 291 methionine, No. 313 threonine, No. 372 arginine, No. 375 lysine, No. 403 glutamine, No. 40 4th Asparagine, No. 2 Examples include 406th serine, 407th isoleucine, and 479th glutamic acid.
- the mutant luciferase described in (c) above is a mature protein from which the secretory signal peptide of the amino acid sequence CLuc of the mutant luciferase described in (a) is excluded.
- mutant luciferase described in (d) above in the fifth mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- the sixth mutant luciferase according to the present invention is any one of the following proteins (a) to (hereinafter referred to as “sixth mutant luciferase”).
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 406th amino acid, and the light emission is 460 nm or more.
- a protein having spectral peak luciferase activity
- the luminescence is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added at a position other than the 406th amino acid, and the light emission is 460 nm or more. Protein with spectral peak luciferase activity.
- the mutant luciferase described in (a) above in the sixth mutant luciferase is a protein comprising an amino acid sequence in which the 406th serine is substituted with another amino acid in the CLuc amino acid sequence.
- the protein exhibits luciferase activity like CLuc.
- this amino acid substitution causes the protein to emit light during luciferin oxidation due to luciferase activity, whereas the emission spectrum peak due to CLuc is 454 nm, whereas it is 460 nm or more, particularly 460 nm to 490 nm (for example, 460 ⁇ ).
- the other amino acid may be any amino acid other than serine, and examples thereof include leucine and isoleucine, and leucine is particularly desirable.
- the mutant luciferase described in (b) above is the mutant luciferase described in (a), and in the mutant luciferase described in (a), one or more (for example, at a position other than the 406th amino acid) (1 to 10, preferably 1 to 5, particularly preferably 1 to 3) amino acid sequence having an amino acid deleted, substituted or added and having a luminescence spectrum peak of 460 nm or more. It is what you have.
- positions other than the 406th amino acid include the 167th threonine, the 375th lysine, the 403th dartamine, the 404th asparagine, the 405th threonine, and the 407th isoleucine. Is mentioned.
- mutant luciferase described in (c) above in the sixth mutant luciferase is: a) A mature protein from which the secretory signal peptide of CLuc is excluded.
- the seventh mutant luciferase according to the present invention is any one of the following proteins (a) to (hereinafter referred to as “seventh mutant luciferase”).
- the mutant luciferase described in (a) above in the seventh mutant luciferase is a protein comprising an amino acid sequence in which the 407th isoleucine is substituted with another amino acid in the CLuc amino acid sequence.
- the protein exhibits luciferase activity like CLuc. However, due to this amino acid substitution, the protein emits light during luciferin oxidation due to luciferase activity!
- the emission spectrum peak by CLuc is 454 ⁇ m, whereas it is 460 nm or more, particularly 460 nm to 490 nm ( For example, the emission spectrum peak is 460 ⁇ ! ⁇ 462nm).
- the other amino acid may be any amino acid other than isoleucine, and examples thereof include glycine and alanine, and alanine is particularly desirable.
- the mutant luciferase described in (b) above in the mutant luciferase described in (a), one or more (for example, 1 to 10, preferably 1 to 5, particularly preferably 1 to 3) amino acids at positions other than the 407th amino acid. It consists of an amino acid sequence in which an acid is deleted, substituted, or added, and has a luminescent spectrum peak luciferase activity of 460 nm or more. Examples of positions other than the 407th amino acid include 167th threonine, 375th lysine, 403th dartamine, 404th asparagine, 405th threonine, and 406th serine. Are listed.
- mutant luciferase described in (c) above in the seventh mutant luciferase is a mature protein excluding the secretory signal peptide of the amino acid sequence ability CLuc of the mutant luciferase described in (a).
- mutant luciferase described in (d) above in the seventh mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- Each of the predetermined amino acid substitutions in the first and third to seventh mutant luciferases and amino acid substitutions at other positions in the amino acid sequence represented by SEQ ID NO: 2 are more than 2 (for example, 2 to 10, Preferably 2 to 8, particularly preferably 2 to 6), and has a luciferase activity with an emission spectrum peak of 458 nm or more, especially 458 nm to 490 nm (for example, 458 ⁇ ! To 475 nm) Proteins are also included in the variant luciferase according to the present invention.
- mutant luciferases containing multiple amino acid substitutions include the following eighth mutant luciferase (hereinafter referred to as ⁇ eighth mutant luciferase '' represented by any one of the following proteins (a) to (: "), U)):
- amino acid sequence of (a) above it consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the above amino acids, and has an emission spectrum peak of 466 nm or more.
- a protein having luciferase activity (c) In the amino acid sequence shown in SEQ ID NO: 2, the 1st to 18th amino acids are deleted, and the 191st leucine, the 235th glutamine, the 280th tyrosine, the 372nd A protein consisting of an amino acid sequence in which arginine, 403th glutamine, 404th asparagine and 405th threonine are replaced with other amino acids;
- amino acid sequence of (c) above consists of an amino acid sequence in which one or several amino acids have been deleted, substituted or added at positions other than the above amino acids, and has an emission spectrum peak of 466 nm or more.
- a protein having luciferase activity is also included in the amino acid sequence of (c) above.
- the mutant luciferase described in (a) above in the eighth mutant luciferase is the 404th asparagine corresponding to the position of the amino acid substitution of the fourth mutant luciferase and the fifth mutant in the CLuc amino acid sequence.
- the 191st leucine, the 235th dartamine, the 280th tyrosine, the 372rd arginine and the 403th glutamine are other amino acids. It is a protein that also has an amino acid sequence ability substituted for. The protein exhibits luciferase activity similar to CLuc.
- the protein emits light at the time of luciferase activity due to luciferase activity, whereas the emission spectrum peak due to CLuc is 454 nm, whereas 466 nm or more, particularly 466 ⁇ ! An emission spectrum peak of ⁇ 490 nm (eg, 466 ⁇ ! To 475 nm) is obtained.
- amino acid substitution at each amino acid position include the following combinations (A) to (G):
- the mutant luciferase described in (b) above is further one or several in the mutant luciferase described in (a) at a position other than the predetermined amino acid described above (E.g. 1-10, preferably 1-5, particularly preferably 1-3) It consists of an amino acid sequence in which an acid has been deleted, substituted or added, and has a luminescence spectrum peak luciferase activity of 466 nm or more.
- the positions other than the above-mentioned predetermined amino acid include the 112th aspartic acid, the 291st methionine, and the 313th threonine.
- mutant luciferase described in (c) above in the eighth mutant luciferase is a mature protein excluding the secretory signal peptide of the amino acid sequence ability CLuc of the mutant luciferase described in (a).
- mutant luciferase described in (d) above in the eighth mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- any two or more of amino acid substitutions in the second mutant luciferase and amino acid substitutions at other positions of the amino acid sequence shown in SEQ ID NO: 2 (for example, 2 to 10, preferably 2 to 8)
- mutant luciferases containing multiple amino acid substitutions include, for example, the ninth mutant luciferase (hereinafter referred to as ⁇ 9th mutant luciferase '' represented by any one of the following proteins (a) to (: And)):
- the mutant luciferase described in (a) above together with the 178th methionine corresponding to the amino acid substitution position of the second mutant luciferase in the amino acid sequence of CLuc, the 191st Leucine, 280th tyrosine, 372rd arginine, 403th glutamine, 404th asparagine, and 405th threonine are substituted with other amino acids.
- the protein exhibits luciferase activity like CLuc.
- the protein emits light at the time of luciferase activity due to luciferase activity, whereas the emission spectrum peak due to CLuc is 454 nm, compared to 435 nm or less, particularly 420 ⁇ ! It becomes an emission spectrum peak of ⁇ 435 nm (for example, 425 nm to 435 nm).
- amino acid substitution at each amino acid position include the following combinations (A) to (G):
- the mutant luciferase described in (b) above is further one or several in the mutant luciferase described in (a) at a position other than the predetermined amino acid described above ( (For example, 1-10, preferably 1-5, particularly preferably 1-3) amino acid sequence having an amino acid deleted, substituted, or added, and having a luminescence spectrum peak of 435 nm or less. It is what has.
- the positions other than the predetermined amino acid described above include the 291st methionine and the 313th threonine.
- mutant luciferase described in (c) above in the ninth mutant luciferase is a mature protein from which the secretory signal peptide of the amino acid sequence ability CLuc of the mutant luciferase described in (a) is excluded.
- mutant luciferase described in (d) above in the ninth mutant luciferase is a mature protein obtained by removing the CLuc secretion signal peptide from the amino acid sequence of the mutant luciferase described in (b).
- each predetermined amino acid substitution is maintained for the amino acid sequence of the protein described in each of (a) or (c) of the first to ninth mutant luciferases described above, and is 80% or more, preferably 90%.
- the mutant luciferase according to the present invention also includes a protein having an amino acid sequence ability having amino acid identity of not less than%, particularly preferably not less than 95%, and having a luciferase activity having a predetermined emission spectrum peak.
- an error may occur in the emission spectrum peak of luciferase due to a measurement method, a spectrum correction method, a smoothing process, or the like. Therefore, as long as the relative emission spectrum peak shift described above with respect to wild-type luciferase (CLuc) is involved, the emission spectrum peak value is within the error range of a numerical value nm (for example, 5 nm, preferably A mutant luciferase having a luciferase activity with an emission spectrum peak of 4 nm, particularly preferably 2 nm, is also included in the mutant luciferase according to the present invention.
- nm for example, 5 nm, preferably A mutant luciferase having a luciferase activity with an emission spectrum peak of 4 nm, particularly preferably 2 nm
- mutant luciferase [0123]
- mutant luciferase the first to ninth mutant luciferases are collectively referred to as "mutant luciferase according to the present invention”.
- the above-described mutant luciferase according to the present invention can be a fusion protein linked to a foreign protein or peptide.
- the foreign protein or peptide means a protein or peptide exogenous to the mutant luciferase according to the present invention.
- proteins or peptides used for protein purification include proteins or peptides used for protein purification (glutathione S-transferase, maltose binding protein, thioredoxin, cellulose binding domain, streptavidin binding peptide, histidine tag, etc.), extracellular secretion or intracellular Signal peptide for translocation to organ Saccharomyces cerevisiae ⁇ - factor secretion signal peptide (amino acid sequence: SEQ ID NO: 3), budding yeast invertase signal peptide (amino acid sequence: SEQ ID NO: 4), budding yeast membrane protein Ste6p signal peptide (amino acid sequence: SEQ ID NO: 5) and the like.
- proteins or peptides used for protein purification glutase, maltose binding protein, thioredoxin, cellulose binding domain, streptavidin binding peptide, histidine tag, etc.
- a gene encoding a fusion protein in which a secretory signal peptide suitable for the host to be transformed and the mature protein of the mutant luciferase according to the present invention is linked is transformed into the host.
- Mutant luciferase can be secreted and expressed extracellularly.
- the position where the foreign protein or peptide is linked to the mutant luciferase according to the present invention can be appropriately selected so that the mutant luciferase according to the present invention and the foreign protein or peptide have their respective functions or activities. .
- the secretory signal peptide is shown on the N-terminal side of the mature protein (that is, shown in SEQ ID NO: 2). (N-terminal side of the 19th amino acid in the amino acid sequence).
- the gene according to the present invention is a gene encoding the mutant luciferase according to the present invention or the above-mentioned fusion protein. By introducing these genes into a host, the mutant luciferase or fusion protein according to the present invention can be expressed.
- the host is not particularly limited, but includes yeast, Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, or Pseudomonas putida. Examples include bacteria belonging to the genus Pseudomonas, animal cells such as COS cells, insect cells such as S19, and plants belonging to the Brassicaceae family.
- the yeast may be any yeast. For example, Saccharomyces cereviche, Schizosaccharomyces pombe, Pichia pastoris, Candida albicans, Hansenula 'Polymorphs (Hansenula polymorpha) are mentioned, and Saccharomyces cereviche is particularly preferable.
- a gene encoding a mutant luciferase according to the present invention or a gene encoding a foreign protein or peptide is prepared.
- These genes can be easily obtained by PCR using primers complementary to the nucleotide sequences at both ends of the region, using, for example, genomic DNA of the organism from which these genes are derived (for example, Cypridina 'noctil force) To gain Can.
- the mutant luciferase according to the present invention has an amino acid substitution in the amino acid sequence of CLuc, the mutation is further introduced into the PCR product obtained as described above by a site-specific mutagenesis method or the like. By doing so, a gene encoding the mutant luciferase according to the present invention can be obtained.
- the DNA fragment having the base sequence is hybridized as a probe by chemical synthesis or by PCR using a cloned probe as a cage.
- a gene encoding the mutant luciferase according to the present invention or a gene encoding a foreign protein or peptide can be obtained.
- a gene encoding the mutant luciferase according to the present invention or a gene encoding a foreign protein or peptide according to the present invention by site-directed mutagenesis or the like, and having a function equivalent to that before the mutation. Can be synthesized.
- a known method such as the Kunkel method or the Gapped dup lex method, or a method analogous thereto can be adopted.
- a mutagenesis kit eg, Mutant-K (TAKARA) or Mutant-G (TAKARA)
- TAKARA's LA PCR in vitr o Mutation is introduced using the Mutagenesis series kit.
- the mutant luciferase according to the present invention is linked to the gene encoding the foreign protein or peptide to produce a gene encoding the fusion protein.
- the mutant luciferase according to the present invention is used.
- Such DNA may be ligated DNA itself or a vector containing the DNA.
- a method for linking a gene encoding a foreign protein or peptide to a gene encoding a mutant luciferase according to the present invention includes a purified gene encoding a mutant luciferase according to the present invention and a foreign protein or peptide, respectively.
- a method is used in which the gene coding for is cleaved with an appropriate restriction enzyme and ligated.
- PCR or the like can be used by providing a region homologous to each of the gene encoding the mutant luciferase according to the present invention and the gene encoding the foreign protein or peptide. It may be a method of linking both by an in vitro method or an in vivo method using yeast or the like.
- a recombinant vector containing the gene according to the present invention can be obtained by inserting the gene according to the present invention into an appropriate vector.
- the vector to be used is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, shuttle vectors, helper plasmids and the like. If the vector itself does not have replication ability, it may be a DNA fragment that can be replicated by inserting it into the host chromosome.
- Plasmid DNA includes plasmids derived from E. coli (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUBll 0, pTP5, etc.), and plasmids derived from yeast (eg, Phylogens include Phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, gtl0, gtll, ⁇ ZAP, etc.).
- animal viruses such as retroviruses or silkworm viruses, and insect virus vectors such as baculoviruses may be used.
- the method of inserting the gene according to the present invention into a vector can be performed according to the above-described method of linking a gene encoding a foreign protein or peptide to the gene encoding the mutant luciferase according to the present invention. .
- a transformant is prepared by introducing a gene according to the present invention or a recombinant vector containing the gene according to the present invention (hereinafter referred to as "recombinant vector according to the present invention") into a host. .
- the method for introducing the recombinant vector according to the present invention into yeast is not particularly limited as long as it is a method for introducing DNA into yeast.
- electroporation electroporation (elect mouth position method), suwe mouth plast method, Examples include the lithium acetate method.
- it may be a method of transforming yeast into a chromosomal replacement using a DNA sequence homologous to an arbitrary region in a vector such as a Yip system or a chromosome.
- the method of introducing the recombinant vector according to the present invention into yeast may be based on various methods described in general experimental documents or academic papers.
- the method of introducing the recombinant vector according to the present invention into bacteria is not particularly limited as long as it is a method of introducing DNA into bacteria.
- a method using calcium ions, an elect mouth position method and the like can be mentioned.
- animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster oocyte cells (CHO cells), mouse L cells, and the like are used.
- the method for introducing the recombinant vector according to the present invention into animal cells include the electopore position method, the calcium phosphate method, and the lipofuxion method.
- insect cells When insect cells are used as hosts, S19 cells and the like are used.
- the method for introducing the recombinant vector according to the present invention into insect cells include the calcium phosphate method, the lipofussion method, and the electopore position method.
- Plant cultured cells When a plant is used as a host, the entire plant body, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) Plant cultured cells are used.
- plant organs eg leaves, petals, stems, roots, seeds, etc.
- plant tissues eg epidermis, phloem, soft tissue, xylem, vascular bundles, etc.
- Plant cultured cells are used.
- Examples of the method for introducing the recombinant vector according to the present invention into a plant include an electroporation method, an agglomerate method, a particle gun method, and a PEG method.
- Confirmation of whether or not the recombinant vector according to the present invention has been incorporated into a host can be performed by PCR, Southern hybridization, Northern hybridization, or the like.
- DNA is prepared from the transformant, PCR is performed by designing DNA-specific primers.
- the amplified product is transformed by performing agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., staining with bromide zyme, SYBR Green solution, etc., and detecting the amplified product as a band. Make sure.
- PCR can be performed using primers previously labeled with a fluorescent dye or the like to detect amplification products.
- a method may be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence, enzyme reaction, or the like.
- the obtained transformant is cultured under conditions that allow it to grow.
- the cultured product or culture supernatant of the transformant is directly used for the measurement of enzyme activity, it is cultured under conditions where the mutant luciferase according to the present invention is not inactivated.
- the temperature is, for example, 4 to 37 ° so that the yeast grows and the mutant luciferase according to the present invention is not inactivated during the culture of the transformed yeast introduced with the recombinant vector or the like according to the present invention.
- C preferably 20-30 ° C.
- the pH of the medium may be set to 3.5 to 6.5, preferably 5.5 to 6.0, for example.
- the culture time is, for example, 1 to 120 hours, preferably the logarithmic growth phase 1 to 24 hours.
- the mutant luciferase according to the present invention or the fusion protein of the mutant luciferase according to the present invention and a foreign protein or peptide can be obtained from the above-mentioned transformant.
- the resulting culture or culture supernatant may cause an enzymatic reaction of the mutant luciferase according to the present invention.
- the substrate luciferin eg, umifire luciferin
- the luciferin derivative for example, the chemical structure of the C2-position, C6-position or C8-position side chain in the imidazopyrazinone skeleton of luciferin, for example, in an aqueous solution of an aromatic, aliphatic, or carboxylic acid amino group, etc.
- the structure and position of the functional group are not limited as long as light is emitted by the sea urchin luciferase. Such substitution can be expected to improve the emission intensity and suppress self-decomposition.
- the condition under which an enzyme reaction occurs means a condition in which a luciferin specifically binds to the active center of the mutant luciferase according to the present invention to form a complex and the enzyme reaction proceeds. Further, the contact means a state in which the mutant luciferase according to the present invention and luciferin in the culture or culture supernatant are close to each other and an enzyme reaction occurs.
- the culture means a culture solution or medium containing the transformant. Mutant luciferase according to the present invention For example, when it is linked to a secretion signal peptide suitable for the host, it is secreted into the medium, so that the culture solution or medium containing the transformant can be used as it is. . Alternatively, a culture supernatant obtained by separating the transformant by centrifugation or the like may be used. The culture supernatant can be subjected to, for example, dilution, concentration, dialysis, purification and the like.
- the temperature is set to, for example, 0 to 40 ° C, preferably 15 to 30 ° C.
- pH 4.0-9.0, for example, Preferably 6.0-8.0.
- the contact time is, for example, 1 second to 30 minutes, preferably 1 second to 30 seconds.
- the pH of the culture or culture supernatant is adjusted to a pH at which the enzyme activity of the mutant luciferase according to the present invention is high. Can be shifted.
- the mutant luciferase according to the present invention is included.
- Tris-HCl buffer Tris-HC1
- pH 3.5 9.0 preferably pH 7.0 8.0
- the concentration of luciferin or a derivative thereof as a substrate relative to the culture or culture supernatant is, for example, the turbidity (for example, absorbance at 600 nm) of the culture or culture supernatant of the transformant having the mutant luciferase according to the present invention.
- the turbidity for example, absorbance at 600 nm
- the measuring method is, for example, using a culture of a transformant or a culture supernatant and a mixture of luciferin or a derivative thereof for luminescence measurement using a luminometer, and measuring enzyme activity as relative luminescence intensity (RLU). To do.
- the turbidity eg, absorbance at 600
- the relative luminescence intensity is divided by the turbidity.
- the calculated value (RLU / OD) can be used as the enzyme activity value.
- the mutant luciferase according to the present invention exhibits luciferase activity with an emission spectrum peak different from that of CLuc.
- the light emission spectrum can be used to distinguish light emission by utilizing the different properties of the emission spectrum, and CLuc can be expressed and corrected by dividing by the light emission derived from the CLuc.
- the transformant grows on an agar medium to form a colony. Therefore, for example, after adding luciferin or a derivative thereof to an agar medium containing a transformant, the enzyme activity can be measured by measuring the luminescence intensity of the colony using, for example, a luminescence detector having a CCD camera or the like. it can.
- the mutant luciferase according to the present invention exhibits a luciferase activity of an emission spectrum peak different from CLuc. Therefore, in addition to the above-mentioned measurement of luciferase activity. For example, using a luminescence detector having a plurality of optical filters having different transmission characteristics and a CCD camera, whether or not the mutant luciferase according to the present invention has a luminescence spectrum peak in the above-mentioned range is measured. .
- the transcriptional activity of a plurality of promoters can be simultaneously evaluated using CLuc and the mutant luciferase according to the present invention as a reporter protein.
- the mutant luciferase according to the present invention and the fusion protein of the wild type CLuc or the mutant luciferase according to the present invention and a foreign protein or peptide, 2 Use the above luciferase.
- the wild type CLuc means the following proteins.
- the fusion protein of wild-type CLuc and a foreign protein or peptide means a fusion protein in which the foreign protein or peptide and the protein (a) or (b) are linked.
- each of these two or more luciferase genes is introduced into the host at the 5 'upstream side with a different promoter linked to each of the evaluation targets.
- the luciferase gene is placed under the control of the promoter.
- the obtained transformant is cultured to obtain a culture or a culture supernatant.
- the silkworm, the culture or the culture supernatant is brought into contact with luciferin or its derivative.
- the transcriptional activity of the plurality of promoters can be simultaneously and quantitatively evaluated by measuring the luminescence intensity due to the difference in the luminescence spectrum peak due to the activities of the introduced luciferases.
- the transcriptional activity of one of the promoters is used as a reference, and the transcriptional activity of other promoters. Can also be corrected.
- the emission intensity based on the difference between a plurality of emission spectrum peaks is, for example, a multi-reporter atsey luminometer “AB-2250 Lumi” manufactured by Atoichi Co., Ltd., which is a device applying the principle described in Japanese Patent Application Laid-Open No. 2004-187652. It can be measured by attaching an appropriate filter set to the “Net Sensor MCA”.
- mutant luciferase according to the present invention or the fusion protein of the mutant luciferase according to the present invention and a foreign protein or peptide is used for BRET (Bioluminance resonance energy transfer) or the like to emit light or release energy. be able to.
- BRET Bioluminance resonance energy transfer
- the mutant luciferase according to the present invention is a fusion protein of the mutant luciferase according to the present invention and a foreign protein or peptide, and luciferin or a derivative thereof. Make contact. This contact oxidizes luciferin to excited oxyluciferin. Next, the excited oxyluciferin and the chemical substance are allowed to act.
- a chemical substance means a substance that can receive excitation energy of a light emitter by energy resonance and emit fluorescence by the energy.
- Examples of chemical substances include fluorescein, FITC, TRITC, TAMRA, and fluorescent proteins such as GFP (green jellyfish-derived green fluorescent protein) and its variants (CFP, YFP, etc.) and DsRed (red-branched red fluorescent protein). Can be mentioned. Further, the action means that oxyluciferin and a chemical substance are arranged at a position where energy can be transferred in distance and phase.
- mutant luciferase according to the present invention it is possible to measure the transcriptional activity (multi-reporter assay) of several different promoters by one luminescence measurement with a single substrate. It is.
- mutant luciferase according to the present invention an emission spectrum that matches the excitation spectrum of a specific chemical substance is provided, a higher BRET efficiency is exhibited, and a strong signal is obtained.
- the mutant luciferase according to the present invention the structural change of a plurality of proteins using a plurality of BRETs can be achieved. Simultaneous analysis is possible.
- a plurality of mutant luciferases according to the present invention and the luciferases derived from them were expressed in Saccharomyces cerevisiae, and their emission spectra were compared.
- the plasmid pCLuRA-TDH3 disclosed in WO 2006/132350 was used as an expression vector for secreting and expressing CLuc.
- This plasmid pCLuRA-TDH3 is a secretory signal peptide of ⁇ -factor of budding yeast
- CLuc amino acid sequence shown in SEQ ID NO: 3
- CLuc mature protein CLuc amino acid sequence shown in SEQ ID NO: 2, excluding the 1-18 amino acid sequence
- a CLuc gene a gene encoding “a CLuc”
- the amino acid sequence shown in SEQ ID NO: 6 is an ⁇ CLuc amino sequence.
- a promoter of Saccharomyces cerevisiae TDH3 (systematic gene name: YGR192C) gene is incorporated upstream (5 'side) of the aCLuc gene.
- This promoter controls the expression of the ⁇ CLuc gene.
- the nucleotide sequence shown in SEQ ID NO: 7 is a partial nucleotide sequence of the plasmid pCLuRA-TDH3, which includes the acluc start codon 5 'upstream 700 bp including the TDH3 promoter sequence, a CLuc coding region, and a CYC 1 terminator sequence. CLuc stop codon 3 'downstream sequence up to 300bp.
- Saccharomyces cerevisiae BY4743 A PRB1 strain was transformed with this plasmid pCLuRA-TDH3.
- an EZ-transformation kit (BIO lOl) was used.
- the obtained transformant was added to a synthetic agar medium (0.67% Yeast nitrogen base without amino acids (Becton Tickinson), 40 ⁇ g / ml athenin, 20 ⁇ g / ml L— containing no uracil.
- a synthetic agar medium 0.67% Yeast nitrogen base without amino acids (Becton Tickinson), 40 ⁇ g / ml athenin, 20 ⁇ g / ml L— containing no uracil.
- Arginine monohydrochloride 100 ⁇ g / ml L-aspartic acid, 100 ⁇ g / ml sodium L-glutamate monohydrate, 20 ⁇ g / ml L-histidine, 60 ⁇ g / ml L-leucine, 30 ⁇ g / ml L-Lysine Hydrochloride, 20 ⁇ g / ml L-Methionine, 50 ⁇ g / ml L-Feralanin, 375 ⁇ g / ml L-Serine, 200 ⁇ g / ml L-Leon , 40 ⁇ g / ml L- ⁇ lippophane, 30 ⁇ g / ml L-tyrosine, 150 ⁇ g / ml L-parin, 2% glucose and 2.0% agar: “SD-ura agar” And cultured at 30 ° C for 3 days.
- a transformant carrying the plasmid 100 ⁇ g
- a transformant having the plasmid pCLuRA-TDH3 obtained as described above was synthesized with a synthetic liquid medium (0.67% Yeast nitrogen base with aqueous amino acids (betatone) having a buffering action and not containing uracil.
- Random point mutations were introduced into the aCLuc coding region of the plasmid pCLuRA-TDH3 using Error Prone PCR.
- the target region into which the mutation was introduced was the first half of the a CLuc coding region (the 900th to 1813th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 7, hereinafter referred to as "N region").
- N region the 900th to 1813th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 7, hereinafter referred to as "N region”.
- the reason for limiting the range is that it is difficult to amplify long regions in Error Prone PCR. This is because there are many cases.
- the reason why the 701st to 899th nucleotide sequences in the nucleotide sequence shown in SEQ ID NO: 7 were not used as the target region was that this region was mostly a region encoding a factor-1 secretory signal peptide. is there.
- mut- CLuc- F ATACTACTATTGCCAGCATTGCTGCTAAAG (SEQ ID NO: 8)
- mut- CLuc- NR2 CACGTGTGAGGCTCGCTCGTCTCCACCCAT (SEQ ID NO: 9)
- the composition of the reaction solution for Error Prone PCR targeting the N region was as follows: Taq DNA polymerase (Ronyu, 1 unit / ⁇ 1) 5 ⁇ 1 ; 10 ⁇ R buffer without magnesium ion 10 ⁇ 1; Deoxynucleotide mixed solution for Error Prone PCR 10 ⁇ 1; 25 mM magnesium chloride 28 ⁇ 1; 5 mM manganese chloride 2.5 ⁇ 1; plasmid pCLuRA-TDH3 solution (150 ng / ⁇ 1) 1 ⁇ 1; m ut- Clue- F (SEQ ID NO: 8) (10 pmol / ⁇ 1) 3 ⁇ 1; mut-CLuc-NR2 (SEQ ID NO: 9) (10 pmol / ⁇ 1) 3 ⁇ 1; sterile water 37.3 ⁇ 1.
- composition of the above-mentioned mixed solution for error prone PCR was as follows: lOOmM dCTP 100 ⁇ 1; lOOmM dTTP 100 ⁇ 1; lOOmM dGTP 20 ⁇ 1; lOOmM dAT P 20 ⁇ 1; Sterile water 760 ⁇ 1.
- PCR was further carried out using the above DNA solution as a saddle (hereinafter referred to as "2nd PCR").
- the composition of the 2nd PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 1 ⁇ 1; 10x KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; mut-Clue- F (SEQ ID NO: 8) (10 pmol / ⁇ 1) 1.5 ⁇ 1; mut-CLuc-NR2 (SEQ ID NO: 9) (lOpmol / ⁇ 1) 1.5 ⁇ 1; the above DNA solution 1 ⁇ 1; Sterile water 33 ⁇ 1.
- 2nd PCR reaction is 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, and 94 ° C A cycle of 15 seconds at 15 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 1 minute at 68 ° C (elongation) was performed in 30 cycles.
- N region fragment As a result of electrophoresis of the PCR product obtained by the 2nd PCR reaction with 1% agarose, a DNA fragment of about 900 bp was confirmed. Hereinafter, this DNA fragment is referred to as “N region fragment”.
- N region fragment solution 201 TE buffer
- Saccharomyces cerevisiae generally undergoes homologous recombination with high probability in cells. Therefore, among the nucleotide sequence of plasmid pCLuRA-TDH3, a linear DNA fragment lacking the nucleotide sequence from 967 to 1703 in the nucleotide sequence shown in SEQ ID NO: 7 (hereinafter referred to as “complement N region”). If the fragment is introduced into Saccharomyces cerevisiae at the same time as the “N region fragment” into which mutations have been introduced as described above, circular DNA (mutant plasmid pCLuRA with mutations introduced into the N region) in Saccharomyces cerevisiae. -THD3) is reconstituted by homologous recombination, and Saccharomyces cerevisiae can be transformed with this reconstituted plasmid.
- complement N region a linear DNA fragment lacking the nucleotide sequence from 967 to 1703 in the nucleotide sequence shown in SEQ ID
- a "complement N region fragment” was prepared by PCR as follows. The following oligo DNA primers were used in PCR.
- vec-CLuc-R GCTTCAGCCTCTCTTTTCTCGAGAG (SEQ ID NO: 10)
- SQ-CLuc-NF2 TTCTCGAGCCGTACAAGGACAGCTGCCGCA (SEQ ID NO: 11)
- the composition of the PCR reaction solution was as follows: KOD plus DNA polymerase (Toyobo) 1 ⁇ 1; 10x KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; vec- CLuc- R (SEQ ID NO: lOXlOpmol / ⁇ 1) 1.5 ⁇ 1; SQ-CLuc-NF2 (SEQ ID NO: l lXlOpmol / ⁇ 1) 1.5 ⁇ 1; plasmid pCLuRA -TDH3 solution (150 ng / ⁇ 1) 1 ⁇ 1; sterilized water 33
- the PCR reaction consisted of one cycle of 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation) and 8 minutes at 68 ° C (annealing and extension). Performed in 30 cycles.
- the overlapping portion between the N region fragment and the complement N region fragment is the nucleotide sequence of the 900th to 966th nucleotides and the 1704th to 1813th nucleotides of the nucleotide sequence shown in SEQ ID NO: 7. It was an array.
- N region fragment solution and complement N region fragment solution were mixed in an amount of 5 ⁇ l each, and Saccharomyces cerevisiae BY4743 A PRB1 strain was transformed by the lithium acetate method. Saccharomyces cerevisiae BY4743 A PRB1 strain used for transformation was smeared on SD-ura agar medium and incubated at 30 ° C for 48 hours. The large number of colonies that appeared were regarded as the N region mutant library.
- a C region mutant library was prepared in which the latter half of the a CLuc coding region was the target region for mutation.
- the target region into which the mutation is introduced is about 60 bp of the latter half of the a CLuc coding region and the 3 ′ non-coding region of the a CLuc coding region (from the 1554th to the 1st position in the nucleotide sequence shown in SEQ ID NO: 7).
- the 2663th nucleotide sequence hereinafter referred to as “C region”.
- the reason for including the 3 'non-coding region of the oc CLuc coding region in the C region is that mutations should be introduced a Intracellular homologous recombination outside the CLuc coding region, and a C-terminal of the CLuc coding region This is so as not to affect the efficiency of variation introduction to the code area.
- PCR was performed on the C region fragment corresponding to the N region fragment of the above N region mutant library in the same manner as the above Error Prone PCR and 2nd PCR except that the following oligo DNA primers were used. Made.
- mut-CLuc-CFl TCTCTGGCCTCTGTGGAGATCTTAAAATGA (SEQ ID NO: 12)
- mut-CLuc-R AACTCCTTCCTTTTCGGTTAGAGCGGATGT (SEQ ID NO: 13)
- a complement C region fragment corresponding to the complement N region fragment of the N region mutant library described above was prepared.
- the complement C region fragment was prepared according to the method for preparing the complement N region fragment. The difference is The oligo DNA primers used and the PCR reaction conditions.
- oligo DNA primers were as follows.
- vec-CLuc-F TCTAGAGGGCCGCATCATGTAATTA (SEQ ID NO: 14)
- SQ-CLuc-CRl TGGACAACCGTCAAACTCCTGGTTGATCTT (SEQ ID NO: 15)
- PCR reaction was 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) for 1 cycle, 94 ° C for 15 seconds (denaturation), 55 ° C for 30 seconds A cycle of seconds (annealing) and 8 minutes (elongation) at 68 ° C was performed in 30 cycles.
- the overlapping portion between the C region fragment and the complement C region fragment is the nucleotide sequence shown in SEQ ID NO: 7 from the 1554th to 1663th nucleotide sequence and from the 2576th to 2663rd The base sequence.
- Saccharomyces cerevisiae BY4743 A PRB1 strain was transformed by the lithium acetate method. Saccharomyces cerevisiae BY4743 A PRB1 strain used for transformation was smeared on SD-ura agar medium and incubated at 30 ° C for 48 hours. A large number of colonies that appeared were regarded as the C region mutant library.
- K375R mutant CLuc represents a mutant CLuc in which lysine corresponding to the 375th position in SEQ ID NO: 2 is substituted with arginine.
- the alphabetical symbol for amino acids is a one-letter code for amino acids according to the recommendations of the International Union of Applied Chemistry and Applied Biochemistry (IUPAC-IUB).
- the “K375R mutant” refers to a clone having the K375R mutant CLuc.
- the plasmid carried by the K375R mutant is designated as “pCLuRA-TDH3 [ K375R] ”.
- mutant CLuc, mutant (clone) having mutant CLuc, and plasmid carried by the mutant are referred to in the same manner.
- Each of the following transformed yeasts (a) to () was cultured with shaking in a buffered SD-ura medium, and then the culture supernatant was collected by centrifugation. It was concentrated about 10 times with a molecular weight of 10,000 (Sartorius).
- composition of the reaction solution was as follows: 1 ⁇ luciferin, lOOmM Tris-HC1, pH 7.5
- FIG. Figure 1 shows the relative emission intensity versus wavelength for each luciferase.
- Wild type is wild type CLuc
- M178K is M178K mutant C
- K375R is the measurement result of K375R mutant CLuc
- K375E is the measurement result of K375E mutant CLuc.
- the emission spectrum peak of wild-type CLuc was 453 nm
- the emission spectrum peak of K375R mutant CLuc was 461 nm, shifted to the longer wavelength side of 8 nm. It was.
- the emission spectrum peak of K375E mutant CLuc was 460 nm, shifted to the longer wavelength side by 7 nm.
- the emission spectrum peak of M178K mutant CLuc was 447 nm, which was shifted to the short wavelength side of 6 nm.
- FFT Fast Fourier Transform
- a wavelength distribution in OriginPr 0, the horizontal axis is regarded as a frequency, so internal processing is performed in Hz.
- LPF low-pass' filter
- data was filtered.
- the filtering period wavelength 0.05 was adopted for all data from the viewpoint of consistency between the original data including noise and the processed data. All data were uniformly cut on OriginPro using the LPF process with a period wavelength component of 0.05 or more, subjected to inverse Fourier transform using the function of the program, and then output to a file. This processing makes it possible to convert to a smooth spectrum curve with less noise without changing the outline and peak position of the spectrum.
- the emission spectrum peaks of wild-type CLuc and other mutant CLuc were also measured from 453 nm to 454 nm (wild-type CLuc), from 461 nm to 463 nm (K375R mutant CLuc), and from 460 nm to 462 nm. It shifted to (K375E mutant CLuc). Since it is considered that the determination of the spectral peak wavelength by visual measurement has a large error, the data processing as described above was applied in a unified manner, and the method of automatically determining the spectral peak wavelength by data processing was used.
- the emission spectrum peak of the variant CLuc was determined based on the method for determining the spectrum peak wavelength.
- T167I mutant (a clone having a T167I mutant C Luc in which the threonine at position 167 was replaced with isoleucine in the amino acid sequence shown in SEQ ID NO: 2; the third mutant luciferase) which corresponds to a transformant having The plasmid retained by this clone is referred to as “pCLuRA-TDH3 [T167I]”.
- the emission spectrum peak due to the mutant CLuc secreted by this T1 671 mutant was 458 nm as a result of measurement by the method described in 2-5 of Example 2, which was shifted to the longer wavelength side by 4 nm compared to the wild type CLuc. It was.
- T167 saturated mutation library a mutant library in which the 167th amino acid is replaced with any one of the other amino acids
- T167 saturation mutation library was constructed as follows.
- the oligo DNA primers used were FAR-F: AACCCT CACTAAAGGGAACAAAAGCTGGCT (SEQ ID NO: 16) and T238- Rev: GTACGGGTTG GCGATGATAGG (SEQ ID NO: 17).
- the DNA fragment obtained by this PCR is In the base sequence shown in SEQ ID NO: 7, it corresponds to the first to 1411th base sequences.
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10 ⁇ KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; FAR- F (SEQ ID NO: 16) (10 pmol / ⁇ 1) 0.6 ⁇ 1; ⁇ 238- Rev (SEQ ID NO: 17) (10 ⁇ mol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuRA-TDH3 solution (3.8 ng / 1) 1 1; Sterilization Water 12.6 ⁇ 1.
- PCR reaction was 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody), and 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 2 at 68 ° C. A cycle of 30 minutes (extension) was performed in 30 cycles.
- the oligo DNA primer used was T238X- Fw: CCTA
- 3 -UTR GTAATACGACTCACTATAGGGCGAA (SEQ ID NO: 19).
- N in the sequence means A, T, G, or C.
- NNN in T238X-Fw (SEQ ID NO: 18) introduces a saturation mutation at the 167th amino acid in SEQ ID NO: 2.
- the DNA fragment obtained by this PCR was identified by the sequence ⁇ NNN '' derived from force T238X-FW (SEQ ID NO: 18), which is the base sequence from the 1st to the 2875th base sequences in the base sequence shown in SEQ ID NO: 7. Random mutation has been introduced into the 3 bases following the 1412th force (codon corresponding to the 167th amino acid in SEQ ID NO: 2).
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ 1; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; T238X— Fw (SEQ ID NO: 18) (10 pmol / ⁇ 1) 0.6 ⁇ 1; 3'-UTR (SEQ ID NO: 19) (10 pmol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuRA-TDH3 solution (3.8 ng / 1) 1 1; Sterile water 12.6 ⁇ 1.
- PCR reaction is 94 ° C for 2 min (inactivation of anti-polymerase antibody) for 1 cycle, and 94 ° C for 15 sec (denaturation), 59 ° C for 30 sec (annealing) and 68 ° C for 2 min A cycle of 30 seconds (extension) was performed in 30 cycles.
- a DNA fragment that can be amplified by this PCR has a base sequence shown in SEQ ID NO: 7 by the sequence “NNN” present in the DNA molecule in force DNA solution B, which is the 900th to 1813th base sequence.
- the third power (the codon corresponding to the 167th amino acid in SEQ ID NO: 2) is randomly introduced.
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 ⁇ 1; 10 ⁇ KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; mut—CLuc — F (SEQ ID NO: 8) (lOpmol / ⁇ l) 1.5 ⁇ 1; mut-CLuc-NR2 (SEQ ID NO: 9) (lOpmol / ⁇ ⁇ ) 1.5 ⁇ ⁇ ; DNA solution A 0.5 ⁇ 1; DNA solution B 0.5 ⁇ 1 ; Sterile water 33 ⁇ 1.
- the PCR reaction consists of 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 53 ° C (annealing) and 1 at 68 ° C.
- the minute (extension) cycle was performed in 30 cycles.
- T167K mutant T167K in which the 167th threonine was substituted with lysine in the amino acid sequence shown in SEQ ID NO: 2.
- the plasmid carried by this mutant is hereinafter referred to as “pCLuRA-TDH3 [T167K]”.
- a mutation was introduced into the ⁇ -factor secreted signal peptide (amino acid sequence: SEQ ID NO: 3) of the aCLuc gene.
- -TDH3 [aP21L, K375R] "was produced.
- the 21st proline shown in SEQ ID NO: 3 and SEQ ID NO: 6 is replaced with leucine (hereinafter referred to as “a P21L mutation”).
- a ⁇ -factor secretion signal peptide having a P21L mutation increases the secretion amount of the protein to be secreted linked to the C-terminal side by 7 times or more (Patent Document 3).
- CLuc encoded in this plasmid is K375R mutant CLuc.
- the plasmid pCLuRA—TDH3 [a P21L] refers to the aCLuc gene of pCLuRA—TDH3 in which the 21st proline shown in SEQ ID NO: 3 and SEQ ID NO: 6 is replaced with leucine. This is a plasmid in which the 762nd base cytosine is replaced with thymine in SEQ ID NO: 7.
- PCR reaction was performed.
- the composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10 ⁇ KOD plus buffer 2 ⁇ ; 2 mM each dNTP mixture 2 ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; mut- CLuc-CFl (SEQ ID NO: 12) (10 pmol/ ⁇ 1) 0.6 ⁇ 1; mut-CLuc- R (SEQ ID NO: 13) (lOpmol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuRA-TD H3 [K375R] solution (lng / 1) 1 ⁇ 1; sterilized water 12.6 PCR reaction consists of 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 53 ° C (annealing) and 1 at 68 ° C. A cycle of minutes 15 seconds (extension) was performed in 30 cycles
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; vec- CLuc -F (SEQ ID NO: 14) (10 pmol / ⁇ 1) 0.6 ⁇ 1; SQ-CLuc-CRl (SEQ ID NO: 15) (lOpmol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuRA-T DH3 [a P21L] solution (lng / ⁇ 1) 1 ⁇ 1; sterilized water 12.6 ⁇ 1.
- the PCR reaction was 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody), and 15 seconds at 94 ° C (denaturation), 30 seconds at 53 ° C (ayling), and 8 at 68 ° C.
- the minute (extension) cycle was performed in 30 cycles.
- DNA fragment contained in each of DNA solution D and DNA solution E consists of the base sequence between 1554th and 1663th and the base between 2576th and 2663th in the base sequence shown in SEQ ID NO: 7. Share the sequence! /
- E. coli DH5a was transformed to form colonies.
- One of the obtained colonies is cultured, and plasmid DNA is extracted and purified by a conventional method.
- the nucleotide sequence between the 1st and 2875th nucleotides in the nucleotide sequence shown in SEQ ID NO: 7 is examined, and the desired nucleotide substitution is performed.
- a new mutant CLuc gene library was prepared by the method described in Example 2.
- pCLuRA-TDH3 [aP21L] was used in place of plasmid pCLuRA-TDH3 as a PCR variant.
- the 405th threonine was replaced with isoleucine.
- a clone having a variant CLuc (corresponding to a transformant having the fifth mutant luciferase) was obtained.
- T405I mutant CLuc As a result of measuring the emission spectrum by the method described in Example 2, the emission spectrum peak of T405I mutant CLuc was 458 nm, which was shifted to the longer wavelength side by 4 nm compared to wild-type CLuc.
- the plasmid carried by this T405I mutant is hereinafter referred to as “pCLuRA-TDH3 [a P21L, T405 1]”.
- a plasmid ⁇ p CLuRA- TDH3 which has a histidine tag at the C-terminus and contains a P21L mutation (X CLuc (SEQ ID NO: 20)) a P21L,-(GS) 3H6] "was constructed.
- SEQ ID NO: 23 is a partial base sequence of the plasmid pCLuRA-TDH3 [aP21L,-(GS) 3H6].
- PCR was performed using pCLuRA-TDH3 as a saddle type.
- the oligo DNA primers used were CLuc (GS) 3H6-F: CACCACCATCACCACCATTAGTCTAGAGGGCCGCAT CATGTAATT (SEQ ID NO: 21) and CLuc (GS) 3H6-R: AGAACCAGAACCAGAACCTTT GCATTCATCTGGTACTTCTAGGGT (SEQ ID NO: 22).
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 ⁇ 1; 10 ⁇ KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; CLuc (GS ) 3H6-F (SEQ ID NO: 21) (10 pmol/ ⁇ 1) 1.5 ⁇ 1; CLuc (GS) 3H6-R (SEQ ID NO: 22) (10 pmol/ ⁇ 1) 1.5 ⁇ 1; Plasmid pCLuRA-TDH3 solution (10ng / ⁇ ⁇ ) 0.1 ⁇ ⁇ ; Sterilized water 34 ⁇ 1.
- PCR reaction is 94 ° C for 2 minutes (anti-polymerase antibody inactivation) for 1 cycle, and 94 ° C for 15 seconds (denaturation), 48 ° C for 30 seconds (annealing), and 68 ° C for 8 minutes ( (Elongation) cycle was performed in 30 cycles.
- both 5 'ends of the obtained DNA fragment were phosphorylated with T4 polynucleotide kinase. This was ligated with T4 DNA ligase as a DNA substrate and circularized. Escherichia coli DH5a was transformed with the circularized DNA. Conventional method from transformed E. coli The plasmid was extracted and purified. This plasmid was double digested with EcoRI and Xbal, and the digest was separated by agarose gel electrophoresis. Next, an approximately l.lkbp fragment containing the region encoding the histidine tag was purified by Sigma GeneElute MINUS EtBr SPIN COLUMNS and ethanol precipitation (DNA fragment G).
- pCLuRA-TDH3 [aP21L] was double-digested with EcoRI and Xbal, and the digests were separated by agarose gel electrophoresis. An approximately 6.5 kbp fragment was similarly purified (DNA fragment H).
- DNA fragment G and DNA fragment H were ligated with T4 DNA ligase using DNA fragment G as a DNA substrate, and Escherichia coli DH5a was transformed with it.
- the transformed Escherichia coli force was extracted and purified by a conventional method.
- the obtained plasmid was examined for the nucleotide sequence (from the first to the 2875th sequence in SEQ ID NO: 23) and confirmed to be the desired nucleotide sequence, and pCLuRA— TDH3 [a P21L, — (GS) 3H6].
- pCLuRA-TDH3 and pCLuRA-TDH3 [a P21L,-(GS) 3H6] were used to transform Saccharomyces cerevisiae BY4743 A PRB1 strain, CLuc and CLuc to which a histidine tag was added were secreted, and the respective emission spectra were measured according to the method described in Example 2. As a result, no difference in the emission spectra was observed between the two. In other words, it was confirmed that there was no difference in the emission spectrum depending on the presence or absence of the histidine tag.
- Example 7 A group of mutant CLuc in which lysine corresponding to position 375 in SEQ ID NO: 2 is substituted with another amino acid
- Example 2 when lysine corresponding to the 375th position in SEQ ID NO: 2 is substituted with arginine or glutamic acid, the emission spectrum peak shifts to the longer wavelength side. Therefore, a group of plasmids for secreting and expressing a group of mutant (and wild type) CLuc, which is one of 20 types of amino acids constituting the normal protein of the 375th amino acid, are as follows: -2 and 7-3.
- the oligo DNA primers used were K446X-F: TGAAGTAGAGAAAGTACGAATCAGG NNNCAATCGACTGTAGTAGTAGAACTCA (SEQ ID NO: 24) and mut-CLuc-R (SEQ ID NO: 13).
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; K446X-F (SEQ ID NO: 24) (10 pmol / ⁇ 1) 0.6 ⁇ 1; mut-CLuc- R (SEQ ID NO: 13) (lOpmol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuRA-TDH3 [a P21L,-(GS) 3H6] solution (lng / 1) 1 ⁇ 1; sterilized water 12.6 1.
- PCR reaction is 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 45 ° C (annealing) and 1 minute at 68 ° C 30 A second (extension) cycle was performed in 30 cycles.
- the oligo DNA primers used were K446-R: CCTGATTCGTACTTTCTCTACTTCA (SEQ ID NO: 25) and mut-CLuc-F (SEQ ID NO: 8).
- the composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10 ⁇ KOD plus buffer 2 ⁇ ⁇ ; 2 ⁇ M each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; K446— R (SEQ ID NO: 25) (lOpmol / ⁇ 1) 0.6 ⁇ 1; mut-CLuc- F (SEQ ID NO: 8) (lOpmol / ⁇ 1) 0.6 ⁇ 1; Plasmid pCLuR A- TDH3 [a P21L,-(GS) 3H6 ] Solution (lng / 1) 1 1; Sterile water 14.6 ⁇ 1.
- PCR reaction is 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) and 15 seconds at 94 ° C (denaturation), 30 seconds at 45 ° C (annealing) and 1 minute at 68 ° C 30 A second (extension) cycle was performed in 30 cycles.
- the oligo DNA primers used were mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-R: (SEQ ID NO: 13).
- the composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 ⁇ ⁇ ; 10 ⁇ KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; mut-CLuc- F (SEQ ID NO: 8) (lOpmol / ⁇ 1) 1.5 ⁇ 1; mut-CLuc-R (SEQ ID NO: 13) (lOpmol / 1) 1.5 1; DNA solution J 1 ⁇ 1; DNA solution ⁇ 1 ⁇ 1; Sterilization Water 33 1.
- ⁇ CR reaction is 2 minutes at 94 ° C (inactivation of anti-polymerase antibody) for 1 cycle, 15 seconds at 94 ° C (denaturation), 30 seconds at 50 ° C (annealing) and 2 minutes at 68 ° C A cycle of 20 seconds (extension) was performed in 30 cycles.
- the oligo DNA primers used were vec-CLuc-F (SEQ ID NO: 14) and vec-CLuc-R: (SEQ ID NO: 10).
- the composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 1 ⁇ ⁇ ; 10x KOD plus buffer 5 ⁇ 1; 2 mM each dNTP mixture 5 ⁇ 1; 25 mM magnesium sulfate 2 1; vec- CLuc- F (SEQ ID NO: 14) (lOpmol / ⁇ 1) 1.5 ⁇ 1; vec- CLuc- R (SEQ ID NO: 10) (lOpmol / ⁇ l) 1.5 ⁇ 1; Plasmid pCLuRA-TDH3 [a P21L,-(GS) 3H6] Solution (lng / 1) 1 ⁇ 1;
- the PCR reaction consists of 2 cycles at 94 ° C (inactivation of anti-polymerase antibody) for 1 cycle and 15 seconds at 94 ° C (
- Saccharomyces cerevisiae BY4743 A PRB1 strain was transformed with an equal mixture of DNA solution L and DNA solution M to form colonies. 96 colonies obtained, Each was cultured in a buffered SD_ura liquid medium, and DNA containing the plasmid was extracted and purified from each. Escherichia coli DH5a was transformed with these DNA samples, and plasmid DNA was extracted and purified according to a conventional method such as the transformant strength of the resulting Escherichia coli, and the nucleotide sequence was examined.
- a plasmid having a codon encoding the amino acid corresponding to the 375th amino acid in SEQ ID NO: 2 was obtained as a codon encoding the following amino acids: alanine, cysteine, aspartic acid, glutamic acid, glycine, Isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, norin, tryptophan and tyrosine.
- Respective plasmids in order are “pCLuRA-TDH3 [a P21L, K375A,-(GS) 3H 6]”, r p CLuRA-TDH3 [a P21L, K375C,-(GS) 3H6] j, “pCLuRA— TDH3 [ ⁇ P21L, K375 D, — (GS) 3H6] ”, r p CLuRA-TDH3 [a P21L, K375E,-(GS) 3H6] j,“ pCLuRA—TDH3 [a P21L, K375G,-(GS) 3H6] J, "PCLuRA- TDH3 [a P21L, K375I,-(GS) 3H6] j," pCLuRA-TDH3 [a P21L, K375K,-(GS) 3H6] J, "pCLuRA- TDH3 [a P21L, K375L,-(GS) 3H6] j, “pCL
- Codon power encoding the amino acid corresponding to the 375th amino acid in SEQ ID NO: 2 pCLuRA-TDH3 [a P21L.K37 5F,-(GS) 3H6], which is a codon encoding phenylalanine, is changed as follows. Made.
- PCR was performed using pCLuRA-TDH3 [a P21L,-(GS) 3H6] as a saddle type.
- the oligo DNA primers used were K446F: TTTCAATCGACTGTAGTAGAACTCA (SEQ ID NO: 26) and K44 6-R: CCTGATTCGTACTTTCTCTACTTCA (SEQ ID NO: 25).
- composition of this PCR reaction solution was as follows: KOD plus DNA polymerase 0.4 ⁇ 1; 10x KOD plus buffer 2 ⁇ ⁇ ; 2 mM each dNTP mixture 2 ⁇ ⁇ ; 25 mM magnesium sulfate 0.8 ⁇ 1; K446F ( SEQ ID NO: 26) (lOpmol / ⁇ 1) 0.6 ⁇ 1; ⁇ 446-R (SEQ ID NO: 25) (lOpmol / ⁇ 1) 0.6 ⁇ 1; Rasmid pCLuRA-TDH3 [aP21L,-(GS) 3H6] solution (lng / 1) 1 1; sterilized water 12.6 ⁇ 1.
- PCR reaction was 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody), and 15 seconds at 94 ° C (denaturation), 30 seconds at 48 ° C (annealing), and 8 at 68 ° C.
- the minute (extension) cycle was performed in 30 cycles.
- both 5 'ends of the obtained DNA fragment were phosphorylated with T4 polynucleotide kinase.
- Escherichia coli DH5a was transformed with the circularized DNA.
- the plasmid was extracted and purified from the transformed Escherichia coli by a conventional method. This plasmid was double digested with BamHI and Xbal, and the digest was separated by agarose gel electrophoresis.
- a fragment of about 2.6 kbp including a region coding for a CLuc in which the codon encoding the amino acid corresponding to the 375th amino acid in SEQ ID NO: 2 was changed was purified by Sigma GeneElute MINUS EtBr SPIN COLUMNS and ethanol precipitation ( DNA fragment N).
- pCLuRA-TDH3 [aP21L,-(GS) 3H6] was double digested with BamHI and Xbal, and the digests were separated by agarose gel electrophoresis. About 5kbp fragment was purified in the same way (DNA fragment P)
- DNA fragment N and DNA fragment P were ligated with T4 DNA ligase using DNA fragment N as a DNA substrate, and Escherichia coli DH5a was used for transformation.
- the transformed Escherichia coli force was extracted and purified by a conventional method.
- the nucleotide sequence of the obtained plasmid (sequence No. 23 in SEQ ID NO: 23 from the 1st to the 2875th) was examined to confirm that the desired nucleotide sequence was obtained, and pCLuRA-TDH3 [a P21L, K375F ,-(GS) 3H6].
- a plasmid substituted with a codon encoding histidine or proline encoding the amino acid corresponding to the 375th amino acid in SEQ ID NO: 2 ("pCLuRA-TD H3 [a P21L, K375H,-(GS ) 3H6] ”and“ pCLuRA-TDH3 [a P21L, K375P,-(GS) 3H6] j) ”, which were prepared using pCLuRA- except that the oligo DNA primers for PCR were different. The method was the same as that for TDH3 [aP21L, K375F,-(GS) 3H6].
- the oligo DNA primers used for the preparation of CLuRA- TDH3 [a P21L, K375H,-(GS) 3H6] are K446H: CATCAATCGACTGTAGTAGAACTCA (SEQ ID NO: 27) and K446-R (SEQ ID NO: 25), while pCLuRA-
- the oligo DNA primers used for the preparation of TDH3 [a P21L, K375P,-(GS) 3H6] were K446P: CCACAATCGACTGTAGTAGAACTCA (SEQ ID NO: 28) and K446-R (SEQ ID NO: 25).
- Saccharomyces cerevisiae BY4743 ⁇ PRB1 strain was transformed with each of the 20 plasmids obtained in 7-2 and 7-3 above.
- Saccharomyces transformed by the plasmid pCLuRA-TDH3 [a P21L, K375K,-(GS) 3H6] 'Clep secreted by cerevisiae is a wild-type CLuc; Saccharomyces transformed with 19 other plasmids' CLuc secreted by cereleche is a mutant CLuc.
- Each was cultured by the method described in Example 2, and the luminescence spectrum was measured using the culture supernatant.
- Table 1 shows the maximum wavelengths of the emission spectra of wild-type CLuc and mutant CLuc.
- K375Y 457 As shown in Table 1, surprisingly, the emission spectrum maximum wavelength given by wild-type CLuc was 454 nm, whereas the other mutant CLuc all showed emission spectra of 457 nm or more. It was the maximum wavelength.
- all of the mutant CLuc listed in Table 1 are the first mutant luciferases (K375A mutant CLuc, K375C mutant CLuc, K375D mutant CLuc, K3 75E mutant CLuc, K375F mutant CLuc, K375G mutation CLuc, K375H mutant CLuc, K3 751 mutant CLuc, K375L mutant CLuc, K375M mutant CLuc, K375N mutant CLuc, K3 75P mutant CLuc, K375Q mutant CLuc, K375R mutant CLuc, K375S mutant CLuc, (K3 75T mutant CLuc, K375V mutant CLuc, K375W mutant CLuc and K375Y mutant CLuc)
- fragment a (475)
- the composition of the PCR reaction solution for amplifying fragment a is as follows: KOD plus DNA polymerase (Toyobo) 0.4 l; pCLuRA-TDH3 plasmid solution (3.8 ng / 1) 1 1; 10 X KOD plu s buffer 2 l; 2 mM each dNTP mixture 2 l; 25 mM magnesium sulfate 0.8 1; FAR— F (SEQ ID NO: 16) 0.6 ⁇ KlOpmol / ⁇ 1); ⁇ 475-rev (SEQ ID NO: 29) 0.6 ⁇ KlOpmol / ⁇ 1); sterilized water 13.6 1.
- PCR reaction 94 ° C for 2 minutes (inactivation of anti-polymerase antibody) 1 cycle, 94 ° C for 15 seconds (denaturation), 49 ° C for 30 seconds (annealing), 68 ° C for 2 minutes 30 seconds
- the (extension) cycle was performed in 30 cycles.
- fragment b (475X) the nucleotide sequence from positions 2102 to 2875 was amplified by PCR.
- this DNA fragment is referred to as “fragment b (475X)”.
- oligo DNA primers were used in PCR for amplifying fragment b (475X): N47 5X-t: tccgtcccgtacagctctcagnnnacttccatctactggcaagat (3rd column 3 ⁇ 4 ⁇ No. DO) and 3 -UTR (SEQ ID NO: 19).
- the composition of the PCR reaction solution when amplifying fragment b (475X) is the same as the composition of the reaction solution when amplifying fragment a (475) except for the primer.
- PCR reaction conditions Only the reaction conditions and annealing temperature when amplifying the piece a (475) were different, and the annealing temperature was 50 ° C.
- fragment a (475) and fragment b (475X) As a result of electrophoresis of the PCR products of the obtained fragment a (475) and fragment b (475X) with 1% agarose, the fragment a (475) of about 2100 bp and the fragment b (475X) of about 800 bp were confirmed. did it. These were mixed, subjected to purification with Sigma GeneElute MINUS EtBr SPIN COLUMNS, phenol extraction, ethanol precipitation, and then dissolved in 101 1 sterile water (fragment a (475), b (475X) mix solution) .
- fragment c (475X) the above fragment a (475), b (475X) mix solution was used as a saddle, overlap PCR was performed, and a long fragment (NNN was substituted for the codon at the target mutation position) In SEQ ID NO: 7, the 1554th force and the 2663th nucleotide sequence) were prepared.
- this DNA fragment is referred to as “fragment c (475X)”.
- oligo DNA primers were used in PCR for amplifying fragment c (475X): mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13).
- the composition of the PCR reaction solution when amplifying fragment c (475X) is as follows: KOD plus DNA polymerase (Toyobo Co., Ltd.) 1 1; Fragment a (475), b (475X) mix solution 1 1 ; 10 X KOD plus buffer 5 l; 2 mM each dNTP mixture 5 1; 25 mM magnesium sulfate 2 l; mut-CLuc-CF1 (SEQ ID NO: 12) 1.5 ⁇ KlOpmol / ⁇ 1); mut-CLuc-R (SEQ ID NO: 13) 1.5 KlOpmol / ⁇ 1); Sterile water 33 ⁇ 1.
- PCR reaction is 1 cycle at 94 ° C for 2 minutes (inactivation of anti-polymerase antibody), 15 seconds at 94 ° C (denaturation), 30 seconds at 61 ° C (annealing), 1 minute at 68 ° C ( (Elongation) cycle was performed in 30 cycles.
- fragment d a linear DNA fragment lacking the 1664th to 2575th positions of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3 was amplified by PCR.
- this DNA fragment is referred to as “fragment d”.
- oligo DNA primers were used in PCR for amplifying fragment d: SQ-CLuc-CR1 (SEQ ID NO: 15) and vec-CLuc-F (SEQ ID NO: 14).
- the composition of the PCR reaction solution when amplifying fragment d differs from the composition of the reaction solution when amplifying fragment c (475X) only in the DNA and oligo DNA primers.
- the following DNA was used as the cocoon type: pCLuRA-TDH3 plasmid solution (3.8 ng / 1) 1 1 1
- the PCR reaction conditions differed from the reaction conditions for amplifying fragment c (475X) only in the annealing temperature and extension time.
- fragment c (475X) As a result of electrophoresis of the obtained PCR products of fragment c (475X) and fragment d with 0.7% agarose, respectively, fragment c (475X) of about llOObp and fragment d of about 7000 bp were confirmed. These were mixed, purified with Sigma GeneElute MINUS EtBr SPIN COLUMNS, extracted with phenol, and precipitated with ethanol, and then dissolved in 101 1 sterile water (fragment c (475X), d mix solution).
- Saccharomyces cerevisiae ⁇ 4743 ⁇ ⁇ 1 was transformed by the lithium acetate method using fragment c (475X), d mix solution 10 ⁇ 1, and smeared on SD-Ura agar medium at 30 ° C. For about 48 hours. Many colonies that appeared were designated as “N404 saturation mutation library”.
- clones considered to have an emission spectrum shift were selected by the same method as 2.3 and 2.4 in Example 2, and emission spectra were further measured.
- the emission spectrum peak of the selected N404G mutant CLuc and N404S mutant CLuc was 458 nm.
- the pCLuRA-TDH3 plasmid in which the amino acid at position 404 in the amino acid sequence shown in SEQ ID NO: 2 has been mutated to asparagine daricin is defined as “pCLuRA-TDH3 [N404G]”.
- fragment a (476)
- oligo DNA primers were used in PCR for amplifying fragment a (476): FAR-F (SEQ ID NO: 16) and T476-rev: gttctgagagctgtacgggac (SEQ ID NO: 31).
- the PCR reaction solution composition when amplifying fragment a (476) differs from the reaction solution composition when amplifying fragment a (475) in Example 8 only in the primer.
- the PCR reaction conditions differed only from the reaction conditions when the fragment a (475) was amplified in Example 8 and the annealing temperature, and annealing was performed at 59 ° C.
- fragment b (476X) the 2105th to 2875th nucleotide sequences were increased by PCR. It was wide. This DNA fragment is hereinafter referred to as “fragment b (476X)”.
- oligo DNA primers were used in the PCR for amplifying fragment b (476X): T47 oX-t w: gtcccgtacagctctcagaacnnntccatctactggcaagatggt (Self sequence number 32) and -UTR (SEQ ID NO: 19).
- the composition of the reaction solution when amplifying fragment b (476X) is different from the reaction solution composition when amplifying fragment b (475X) in Example 8 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment b (475X) was amplified in Example 8.
- Fragments a (476) and b (476X) mix solutions were prepared in the same manner as in Example 8.
- fragment c (47 6X) The above fragment a (476), b (476X) mix solution was used as a saddle, overlap PCR was performed, and one long fragment in which the codon at the target mutation position was replaced with NNN (SEQ ID NO: 7 1554 to 2663 base sequences) were prepared.
- this DNA fragment is referred to as “fragment c (47 6X)”.
- composition of the PCR reaction solution when amplifying fragment c (476X) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used as the vertical DNA: fragment a (476), b (476X) mix solution 11.
- the PCR reaction conditions are the same as the reaction conditions when the fragment c (475X) was amplified in Example 8.
- the emission spectrum peak of the selected T405M mutant CLuc was 457 nm.
- fragment a (477).
- oligo DNA primers were used in PCR to amplify fragment a (477): FAR- F (SEQ ID NO: 16) and S477-rev: agtgttctgagagctgtacgg (SEQ ID NO: 33).
- the PCR reaction solution composition for amplifying fragment a (47 7) differs from the reaction solution composition for amplification of fragment a (475) in Example 8 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment a (475) was amplified in Example 8.
- fragment b (477X) the base sequences from 2108th to 2875th in SEQ ID NO: 7 were amplified by PCR. This fragment is referred to as “fragment b (477X)”.
- oligo DNA primers were used in PCR to amplify fragment b (477X): S47 7X-t: ccgtacagctctcagaacactnnnatctactggcaagatggtgac (self-column number 34) and -UTR, SEQ ID NO: 19).
- the PCR reaction solution composition for amplifying fragment b (477X) is V in Example 8, and only the primer differs from the reaction solution composition for amplifying fragment b (475X).
- the PCT reaction conditions are the same as those in Example 8 when fragment b (475X) was amplified.
- PCR reaction solution composition when amplifying fragment c (477X) is different from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the cage DNA.
- the following DNA was used as the vertical DNA: Fragment a (477), b (477X) mix solution 1 ⁇ 1.
- PCR reaction conditions are the same as in Example 8.
- the emission spectrum peak of the selected S406L mutant CLuc was 460 nm.
- the 407th amino acid is the other amino acid A mutant library substituted with either one was prepared.
- the manufacturing method is the same as in Example 8.
- fragment a (478).
- the following oligo DNA primers were used in PCR for amplifying fragment a (478): FAR-F (SEQ ID NO: 16) and I478-rev: ggaagtgttctgagagctgta (SEQ ID NO: 35).
- the PCR reaction solution composition for amplifying fragment a (478) differs only in the reaction conditions and primers for the amplification of fragment a (475) in Example 8.
- the PCR reaction conditions were different from the reaction mixture composition and annealing temperature at the time of amplification of fragment a (475) in Example 8, and annealing was performed at 55 ° C.
- fragment b (478X) the nucleotide sequence from the 2111st position to the 2875th position in SEQ ID NO: 7 was amplified by PCR. This DNA fragment is referred to as “fragment b (478X)”.
- the following oligo DNA primers were used in PCR to amplify fragment b (478X): I478X-Fw: tacagctctcagaacacttccnnntactggcaagatggtgacata (Toki column number 36) and UTR (Tutami column number 19).
- the PCR reaction solution composition for amplifying fragment b (478X) is different from the reaction solution composition for amplifying fragment b (475X) in Example 8 only in the primer.
- the PCR reaction conditions differed from the reaction conditions when the fragment b (475X) was amplified in Example 8 and the annealing temperature, and annealing was performed at 58 ° C.
- fragment 2 (bp) of about 2100 bp and fragment b (478X) of about 800 bp were confirmed. did it.
- Promega Wizard registered trademark
- SV Gel and PCR Clean-Up system phenol extraction, ethanol precipitation, and then dissolved in 10 1 sterile water (fragment & (478), 1) (478) mix solution) 0
- fragment c (478X) the above fragment a (478), b (478X) mix solution was used as a saddle, overlap PCR was performed, and one long fragment (in which the codon at the target mutation position was replaced with NNN ( In SEQ ID NO: 7, the 1554th force and the 2663th nucleotide sequence) were prepared.
- this DNA fragment is referred to as “fragment c (478X)”.
- the PCR reaction solution composition for amplifying fragment c (478X) was the same as that of fragment c (475X) in Example 8. The only difference is the composition of the reaction solution when the DNA is amplified and the DNA in the cage shape. The following DNA was used for the vertical type: Fragment a (478), b (478X) mix solution 1 ⁇ 1.
- the PCR reaction conditions differed only in the annealing conditions in the reaction conditions when the fragment c (475X) was amplified in Example 8, and annealing was performed at 60 ° C.
- the emission spectrum peak of the selected I407A mutant CLuc was 460 nm.
- Double mutant CLuc (first and third mutant luciferases) in which the 167th amino acid is replaced with threonine strength lysine and the 375th amino acid is replaced with lysine strength arginine in the amino acid sequence shown in SEQ ID NO: 2.
- the DNA coding for was prepared as follows
- fragment a (238) A DNA fragment containing a mutation at the 167th amino acid and having a base sequence ability up to the 1st force and 1663th position in SEQ ID NO: 7 is hereinafter referred to as "fragment a (238)".
- the following oligo DNA primers were used for PCR when amplifying this fragment a (238): FA RF (SEQ ID NO: 16) and SQ-CLuc-CRl (SEQ ID NO: 15).
- the PCR reaction solution composition for amplifying fragment a (238) is the same as the reaction solution composition for amplifying fragment a (475) in Example 8, the amount of sterile water, Different.
- the amount of sterilized water and the vertical DNA used were as follows: sterilized water 12.6 ⁇ 1, P CLuRA-TDH3 [T167K] 1 ⁇ l (4.5 ng / ⁇ 1).
- fragment b (446)
- the following oligo DNA primers were used for PCR when amplifying this fragment b (446): mut-CLuc-CFl (SEQ ID NO: 12) and 3'-UTR (SEQ ID NO: 19).
- the PCR reaction solution composition used to amplify fragment b (446) differs from the reaction solution composition used to amplify fragment a (238) only in the form of DNA and primers.
- the following DNA was used as the cocoon type: pCLuRA-TDH 3 [a P21L, K375R] 1 ⁇ 1 (2. Ong / ⁇ 1).
- the PCR reaction conditions are the same as the reaction conditions when fragment a (238) was amplified.
- fragment c (238,446) This DNA fragment is referred to as “fragment c (238,446)”.
- the following oligo DNA primers were used in PCR for amplifying fragment c (238,446): mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-R (SEQ ID NO: 13).
- the PCR reaction solution composition when amplifying fragment c (238, 446) is the same as the reaction solution composition obtained when amplifying fragment c (475X) in Example 8, and the DNA in the cage shape. And only the primers are different.
- the following DNA was used as the cage DNA: Fragments a (238), b (446) mix solution 11.
- the PCR reaction conditions differed from the reaction conditions when amplifying the fragment c (475X) in Example 8 only in the annealing temperature and the extension time, and the annealing temperature was 60 ° C. and the extension time was 2 minutes.
- fragment d (238,446)
- oligo DNA primers were used for PCR when amplifying this fragment d (238,446): vec-CLuc-R (SEQ ID NO: 10) and vec-CLuc-F (SEQ ID NO: 14).
- the composition of the PCR reaction solution for amplifying fragment d (238,446) is the same as the reaction mixture used when amplifying fragment d in Example 8. Only the primer differs from the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment d was amplified in Example 8.
- Saccharomyces cerevisiae BY4743 ⁇ ⁇ ⁇ strain was transformed with ZYMO RESEARCH Frozen-EZ Yeast Transformation II TM using fragments c (238,446), d (238,446) mix solution 10 ⁇ 1 went.
- the emission spectrum peak of the prepared T167K / K375R double mutant CLuc was 460 nm.
- the pCLuRA-TDH3 plasmid in which the 167th amino acid was mutated from threonine to lysine and the 375th amino acid was mutated from lysine to arginine in the amino acid sequence shown in SEQ ID NO: 2 was designated as ⁇ pCLuRA
- a gene encoding 3 mutant luciferase was prepared. Examples of methods for introducing mutations
- fragment b (474)
- the composition of the PCR reaction solution when amplifying fragment b (474) differs from the reaction solution composition when amplifying fragment b (446) in Example 12 only in the cage DNA.
- the following DNA was used as the ⁇ -type DNA: pCLuRA-TDH3 [Q403P] (The amino acid at the 403rd in the amino acid sequence shown in SEQ ID NO: 2 obtained as a result of the screening described in Example 2 was derived from glutamine. PCLuRA-TDH3 plasmid mutated to proline) 1 ⁇ l (2.56 ng / ⁇ 1).
- the PCR reaction conditions are the same as the reaction conditions when fragment b (446) was amplified in Example 12.
- fragment c (238,474) The above fragment a (238), b (474) mix solution was used as a saddle type to perform overlap PCR, and a single long !! fragment (SEQ ID NO: 7 was substituted with the amino acid at the target position) The nucleotide sequence from the 900th to the 2663th base sequence) was prepared. Hereinafter, this DNA fragment is referred to as “fragment c (238,474)”.
- composition of the PCR reaction solution when amplifying fragment c (238,474) differs from the reaction solution composition when amplifying fragment c (238,446) in Example 12 only in the cage DNA.
- the following DNA was used for the vertical DNA: Fragment a (238), b (474) mix solution 11.
- PCR reaction conditions are the same as in Example 12.
- the emission spectrum of the obtained clone was measured.
- fragment b (475)
- reaction conditions are the same as those in the amplification of fragment b (446) in Example 12.
- fragment c (238,475) a mixed solution of fragments a (238) (Example 12) and b (475) was prepared in the same manner as in Example 12.
- the above fragment a (238), b (475) mix solution was used as a trapezoid to perform overlap PCR, and a single long !, fragment (SEQ ID NO: 7) in which the amino acid at the target position was substituted.
- the 900th to 2663th nucleotide sequences) were prepared.
- this DNA fragment is referred to as “fragment c (238,475)”.
- composition of the PCR reaction solution when amplifying fragment c (238,475) differs from the reaction solution composition when amplifying fragment c (238,446) in Example 12 only in the cage DNA.
- the following DNA was used as the cocoon type: Fragment a (238), b (475) mix solution 1 1.
- PCR reaction conditions are the same as in Example 12.
- the emission spectrum of the obtained clone was measured.
- the emission spectrum peak of the prepared T167K / N404G double mutant CLuc was 460 nm.
- fragment b (476)
- the composition of the PCR reaction solution when amplifying fragment b (476) is different from the reaction solution composition when amplifying fragment b (446) in Example 12 only in the DNA in a cage shape.
- the following DNAs were used as the cocoon type: pCLuRA-TDH3 [aP21L, T405I] (Example 5) 1 ⁇ l (2.0 ng / ⁇ 1).
- the PCR reaction conditions are the same as the reaction conditions when fragment b (446) was amplified in Example 12.
- composition of the PCR reaction solution when amplifying fragment c (238,476) is different from the reaction solution composition when amplifying fragment c (238,446) in Example 12 only in the DNA in a cage shape.
- the following DNA was used for the cage type: Fragments a (238), b (476) mix solution 1 1.
- PCR reaction conditions are the same as in Example 12.
- the emission spectrum of the obtained clone was measured.
- fragment a (268).
- oligo DNA primers were used in PCR for amplifying fragment a (268): FAR-F (SEQ ID NO: 16) and L268-rev: gatgtcgatcacgatcagttt (SEQ ID NO: 37). Further, the PCR reaction solution composition when amplifying fragment a (268) differs from the reaction solution composition when amplifying fragment a (475) in Example 8 only in the primer. The PCR reaction conditions are the same as the reaction conditions when fragment a (475) was amplified in Example 8.
- fragment b (268X)
- L26 8X-Fw aaactgatcgtgatcgacatcnnnggaggaagatctgtaagaatc (self-sequence number 38) and 3 UTR (SEQ ID NO: 19).
- the PCR reaction solution composition for amplifying fragment b (268X) differs from the reaction solution composition for the amplification of fragment b (475X) in Example 8 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment b (475X) was amplified in Example 8.
- fragment c (268X) The above fragment a (268), b (268X) mix solution was used as a saddle type, overlap PCR was performed, and one long fragment in which the codon at the target mutation position was substituted with NNN (SEQ ID NO: 7 The base sequence from the 900th position up to the 1813th position was prepared.
- fragment c (268X) This DNA fragment is referred to as “fragment c (268X)”.
- the following oligo DNA primers were used in PCR for amplifying fragment c (268X): mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-NR2 (SEQ ID NO: 9).
- the PCR reaction solution composition for amplifying fragment c (268X) is the same as the reaction solution composition for amplifying fragment c (475X) in Example 8, and only the DNA and primers in the vertical form. Different.
- the following DNA was used as the vertical DNA: Fragment a (268), b (268X) mix solution 11.
- the PCR reaction conditions differed only from the reaction conditions when the fragment c (475X) was amplified in Example 8 and the annealing temperature, and annealing was performed at 53 ° C.
- fragment d (268). a linear DNA lacking the 967th to 1703th positions of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3 was amplified by PCR. This DNA fragment is referred to as “fragment d (268).”
- the following oligo DNA primers were used in PCR for amplifying fragment d (268): vec-CLuc-R (SEQ ID NO: 10) and SQ-CLuc-NF2 (SEQ ID NO: 11).
- the PCR reaction solution composition for amplifying fragment d (268) differs from the reaction solution composition for amplifying fragment d in Example 8 only in the primer.
- the PCR reaction conditions differed only from the reaction conditions when the fragment d was amplified in Example 8 and the annealing temperature, and annealing was performed at 62 ° C.
- fragment c (268X) and d (268) were electrophoresed with 0.7% agarose, and as a result, approximately 900 bp fragment c (268X) and approximately 7000 bp fragment d (268) were confirmed. did it.
- fragment c (268X) and d (268) mix solutions were prepared in the same manner as in Example 8.
- a gene encoding the double mutant CLuc (second mutant luciferase) of M178K and L197P in the amino acid sequence shown in SEQ ID NO: 2 was prepared.
- the method for introducing the mutation is the same as in Example 12.
- fragment a A DNA fragment containing the mutation at the position of amino acid number 178 shown in SEQ ID NO: 2 and consisting of the nucleotide sequence of the first force in SEQ ID NO: 7 and the 1492th base sequence was amplified by PCR.
- this DNA fragment is referred to as “fragment a (249)”.
- oligo DNA primers were used in PCR for amplifying fragment a (249): FAR-F (SEQ ID NO: 16) and SQ-CLuc-F001-rev: cacgatcagtttgaagaattctatgacggt (SEQ ID NO: 39).
- the PCR reaction solution composition for amplifying fragment a (249) is different from the reaction solution composition for amplifying fragment a (475) in Example 8 only in the cage DNA and primers.
- ⁇ -type DNA pCLuRA- TDH3 [M178K] (Example 2) 1 ⁇ l (2.85 ng / ⁇ PCR reaction conditions were the same as those in the amplification of fragment a (475) in Example 8. Only the conditions and annealing temperature were different, and annealing was performed at 58 ° C.
- fragment b (268) a DNA fragment having the nucleotide sequence from the 1463th position to the 2875th position in SEQ ID NO: 7 containing a mutation at the position of amino acid number 197 shown in SEQ ID NO: 2 was amplified by PCR.
- this DNA fragment is referred to as “fragment b (268)”.
- oligo DNA primers were used for PCR when amplifying this fragment b (268): mut-CLuc-CFO: accgtcatagaattcttcaaactgatcgtg (SEQ ID NO: 40) and 3'-UTR (SEQ ID NO: 19).
- the PCR reaction solution composition used to amplify fragment b (268) differs from the PCR reaction solution composition used to amplify fragment a (249) only in the DNA and primers.
- the DNA used as a saddle type was as follows: pCLuRA-TDH3 [L197P] (16-1 above) 1 ⁇ l (3.53 ng / ⁇ 1). PCR The reaction conditions are the same as the reaction conditions when fragment a (249) was amplified.
- fragments a (249) and fragment b (268) were electrophoresed with 1% agarose to confirm that they were about 1500 bp and about 1400 bp DNA fragments, respectively. These were mixed to prepare fragments a (249) and b (268) mix solutions in the same manner as in Example 8.
- fragment c (249,268)
- composition of the PCR reaction solution when amplifying fragment c (249,268) differs from the reaction solution composition when amplifying fragment c (268 X) in the above 16-1, only in the DNA in the saddle shape.
- the following DNA was used for the vertical type: fragments a (249), b (268) mix solution 11.
- PCR reaction conditions differed from the reaction conditions when fragment c (268X) was amplified in 16_1 above, only in the annealing temperature and extension time, and the annealing temperature was 60 ° C and the extension time was 1 minute 30 seconds.
- the 21st amino acid in the signal sequence of ex CLuc shown in SEQ ID NO: 6 was substituted with leucine.
- the method for introducing the mutation involves the DNA fragment containing the mutation corresponding to the 21st position in the amino acid sequence shown in SEQ ID NO: 6, and the 178th and 197th amino acids in the amino acid sequence shown in SEQ ID NO: 2.
- a DNA fragment containing the two mutations at the position was amplified by PCR and prepared by overlapping PCR using these two DNA fragments. It was.
- fragment a (21) A DNA fragment containing the mutation at the position of amino acid number 21 shown in SEQ ID NO: 6 and consisting of the base sequence of the first force and 966th in SEQ ID NO: 7 was amplified by PCR.
- this DNA fragment is referred to as “fragment a (21)”.
- the composition of the PCR reaction solution when amplifying fragment a (21) differs from the reaction solution composition when amplifying fragment a (238) in Example 12 only in the DNA and primers.
- the following DNA and oligo DNA primers were used: pCLuRA-TDH3 [a P21L] (Patent Document 3) 1 ⁇ l (4.25 ng / ⁇ 1), FAR-F (SEQ ID NO: 16) and vec -CLuc-R (SEQ ID NO: 10).
- the PCR reaction conditions differed only from the reaction conditions when the fragment a (238) was amplified in Example 12 and the annealing temperature, and annealing was performed at 53 ° C.
- fragment b (249,268)
- the PCR reaction solution composition when amplifying fragment b (249,268) differs from the PCR reaction solution composition when fragment a (21) is amplified only in the DNA and primers.
- the following DNA and oligo DNA primers were used: pCLuRA- TDH3 [M178K, L197P] (16-2 above) 1 ⁇ l (1.56ng / ⁇ 1), mut-CLuc-F (SEQ ID NO: 8) And 3'-UTR (SEQ ID NO: 19).
- the PCR reaction conditions are the same as the reaction conditions when fragment a (21) was amplified.
- fragment c (21,249,268)
- the following oligo DNA primers were used in PCR to amplify fragment c (21,249,268): SQ-GPD1-FO: ATGTATCTATCTCATTTTCTTACA (SEQ ID NO: 41) and mut-CLuc-NR2 (SEQ ID NO: 9).
- the PCR reaction solution composition for amplifying fragment c (21,249,268) is In Example 12, the composition of the reaction solution when the fragment c (238,446) was amplified and only the DNA and the primer to be used were different.
- the following DNA was used for the cage type: Fragment a (21), b (249,268) mix solution 1 1.
- the PCR reaction conditions differed only from the reaction conditions when the fragment a (238) was amplified in Example 12 and the annealing temperature, and the annealing temperature was 51 ° C.
- fragment d 21,249,268
- the following oligo DNA primers were used in PCR to amplify fragment d (21,249,268): SQ-GPD1-R0: CAGCTTTTTCCAAATCAGAGAGAGCAG (SEQ ID NO: 42) and S Q-CLuc-NF2 (SEQ ID NO: 11).
- the PCR reaction solution composition for amplifying fragment d (21,249,268) is different from the reaction solution composition for the amplification of fragment d (238,446) in Example 12 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment d (238,446) was amplified in Example 12.
- a gene encoding 1 mutant luciferase was prepared. Examples of methods for introducing mutations
- fragment a (446)
- the following oligo DNA primers were used in PCR to amplify fragment a (446): mut- CLuc-F (SEQ ID NO: 8) and SQ-CLuc-F002-rev: caaccagaatctgttttccatcaa (SEQ ID NO: 43).
- the PCR reaction solution composition for amplifying fragment a (446) is different from the reaction solution composition for amplifying fragment a (238) in Example 12 only in the DNA and primers.
- the following DNA was used as a cage type: pCLuRA-TDH3 [a P21L, K375R] (Example 4) 1 ⁇ 1 (2.0 ⁇ // ⁇ 1).
- the PCR reaction conditions are the same as the reaction conditions when fragment a (238) was amplified in Example 12.
- fragment (474) a DNA fragment having a nucleotide sequence from 2064th to 2875th in SEQ ID NO: 7 containing a mutation at the 403rd amino acid is hereinafter referred to as "fragment (474)".
- the following oligo DNA primers were used for PCR when amplifying this fragment (474): SQ-CLuc-F002: ttgatggaaaacagattctggttg (SEQ ID NO: 44) and 3, -UTR (SEQ ID NO: 19).
- the PCR reaction solution composition for amplifying fragment (474) is different from the reaction solution composition for amplifying fragment b (474) in Example 13 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment b (446) was amplified in Example 12.
- fragment c (446,474) a DNA fragment with the amino acid at the target position (SEQ ID NO: 7 In 1980, the 1554th nucleotide and the 2663th nucleotide sequence) were prepared.
- this DNA fragment is referred to as “fragment c (446,474)”.
- the PCR reaction solution composition when amplifying fragment c (446,474) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used as the cocoon type: Fragment a (446), (474) Mix solution 1 1.
- the PCR reaction conditions are the same as the reaction conditions when fragment c (238,446) was amplified in Example 12.
- fragment (475) The DNA fragment having the nucleotide sequence from 2064th to 2875th in SEQ ID NO: 7 containing the mutation at the 404th amino acid is hereinafter referred to as "fragment (475)”.
- composition of the PCR reaction solution for amplifying the fragment (475) was the same as that of the fragment (474) in Example 17.
- Fragment a (446) of Example 17 and the PCR product of fragment (475) were electrophoresed with 1% agarose to prepare a mixed solution of fragments a (446) and (475) as in Example 12. did.
- fragment c (446,475
- the PCR reaction solution composition when amplifying fragment c (446,475) is different from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the cage DNA.
- the following DNA was used for the vertical type: fragments a (446), (475) mix solution 1 1.
- the PCR reaction conditions are the same as the reaction conditions when fragment c (475X) was amplified in Example 8.
- fragment (476) A DNA fragment comprising the 2064th to 2875th nucleotide sequence in SEQ ID NO: 7 containing the mutation at the 405th amino acid is hereinafter referred to as "fragment (476)”.
- composition of the PCR reaction solution for amplifying fragment (476) was the same as that of fragment (474) in Example 17.
- Fragment a (446), fragment (476) PCR product of Example 17 was electrophoresed with 1% agarose, and in the same manner as Example 17, fragment a (446), (476) mix solution was made.
- fragment c (446,476
- the composition of the PCR reaction solution when amplifying fragment c (446,476) is different from the PCR reaction solution composition of Example 8 only in the DNA in the saddle shape.
- the following DNA was used as the cocoon type: fragments a (446), (476) mix solution 1 1.
- the PCR reaction conditions are the same as the reaction conditions when fragment c (238,446) was amplified in Example 12.
- the emission spectrum peak of the prepared K375R / T405I double mutant CLuc was 463 nm.
- fragment a the DNA fragment consisting of the nucleotide sequence from the first force to the 2119th nucleotide is hereinafter referred to as “fragment a (474)”.
- the PCR reaction solution composition for amplifying fragment a differs from the PCR reaction solution composition for the fragment a (475) in Example 8 only in the primer.
- the PCR reaction conditions differed only from the reaction conditions when the fragment a (475) was amplified in Example 8 and the annealing temperature, and annealing was performed at 55 ° C.
- fragment b (474,475 ) a DNA fragment having a nucleotide sequence from the 2099th position to the 2875th position in SEQ ID NO: 7 containing a mutation at the 403rd and 404th amino acids was referred to as "fragment b (474,475 ) ".
- PCR for amplifying this fragment b (474,475) uses the following oligo DNA primers: Q474P / N475- ⁇ w: gtgtccgtcccgtacagctctcccgggacttccatctactggcaagat ( ⁇ c column 3 ⁇ 4 ⁇ No. 46) and 3'-UTR (SEQ ID NO: 19).
- the PCR reaction solution composition when amplifying fragment b (474,475) differs from the PCR reaction solution composition when amplifying fragment b (475X) in Example 8 only in primers.
- the PCR reaction conditions differed only from the reaction conditions when the fragment b (475X) was amplified in Example 8 and the annealing temperature, and annealing was performed at 58 ° C.
- fragment c (474,475)
- the PCR reaction solution composition when amplifying fragment c (474,475) is different from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used as the cocoon type: fragments a (474), b (474,475) mix solution 11.
- the PCR reaction conditions were the same as those in Example 8 when amplifying the fragment c (475X), annealing temperature and extension time. However, the annealing temperature was 60 ° C and the extension time was 1 minute 30 seconds.
- the emission spectrum peak of the prepared Q403P / N404G double mutant CLuc was 462 nm.
- a gene encoding 5 mutant luciferase was prepared. Examples of methods for introducing mutations
- fragment b (474,476)
- the PCR for amplifying this fragment b (474,476) uses the following oligo DNA primers: Q474P / T476I- J ⁇ W: gtgtccgtcccgtacagctctcccaacatctccatctactggcaagatggt (Tsuki column number 47) and 3'-UTR (SEQ ID NO: 19). Further, the PCR reaction solution composition when amplifying fragment b (474,476) differs from the reaction solution composition when amplifying fragment b (475X) in Example 8 only in the primer. The PCR reaction conditions are the same as the reaction conditions when fragment b (474,475) was amplified in Example 20.
- fragment c (474,476)
- composition of the PCR reaction solution when amplifying fragment c (474,476) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used as the cocoon type: Fragment a (474), b (474,476) mix solution 11.
- PCR reaction conditions are described in Example 20. In this case, the reaction conditions are the same as when fragment c (474,475) was amplified.
- a DNA fragment having a nucleotide sequence from position 2102 to position 2875 in SEQ ID NO: 7 containing a mutation at the 404th and 405th amino acids is hereinafter referred to as "fragment b (475,476)”.
- PCR for amplifying this fragment b (475,476) uses the following oligo DNA primers: 7/5 / l 47oi— rw: tccgtcccgtacagctctcaggggatctccatctactggcaagatggt (3 ⁇ 4c ⁇ lj3 ⁇ 4> ⁇ 48) and 3'-UTR ( SEQ ID NO: 19).
- the PCR reaction solution composition for amplifying fragment b (475,476) differs from the PCR reaction solution composition for the fragment b (475X) in Example 8 only in primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment b (474,475) was amplified in Example 20.
- fragment c (475,476)
- the composition of the PCR reaction solution when amplifying fragment c (475,476) is different from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the type of DNA The following was used: Fragments a (475), b (475,476) mix solution 1 1.
- the PCR reaction conditions are the same as the reaction conditions when fragment c (474,475) was amplified in Example 20.
- a gene encoding the triple mutant C Luc (fourth and fifth mutant luciferases) of Q403P, N404G and T405I in the amino acid sequence shown in SEQ ID NO: 2 was prepared.
- the method for introducing the mutation is the same as in Example 12.
- a DNA fragment comprising the nucleotide sequence from 2099 to 2875 in SEQ ID NO: 7 and containing the mutations at the 403rd, 404th and 405th amino acids is referred to as “fragment b" (474,475,4761) ".
- fragment c (474,475,4761)
- the PCR reaction solution composition when amplifying fragment c (474,475,476I) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used as the cocoon type: Fragment a (474), b (474,475,476I) mix solution 11.
- the PCR reaction conditions are the same as those in the amplification of fragment c (474,475) in Example 20.
- a gene encoding the triple mutant CLuc (fourth and fifth mutant luciferases) of Q403P, N404G and T405M in the amino acid sequence shown in SEQ ID NO: 2 was prepared.
- the method for introducing the mutation is the same as in Example 12.
- a DNA fragment comprising the nucleotide sequence from 2099 to 2875 in SEQ ID NO: 7 and containing the mutations at the 403rd, 404th and 405th amino acids is referred to as "fragment b" (474,475,476M) ".
- the following oligo DNA primers were used in PCR to amplify this fragment b (474,475,476M): Q474P / N475G / T476M-Fw: gtgtccgtcccgtacagctctcccgggatgtccatctactg gcaagatggt (SEQ ID NO: 50) and 3'-UTR (SEQ ID NO: 19).
- the PCR reaction solution composition when amplifying fragment b (474,475,476M) is different from the PCR reaction solution composition when amplifying fragment b (475X) in Example 8 only in the primer.
- the PCR reaction conditions are the same as those in the amplification of fragment b (474,475) in Example 20.
- fragments a (474) (Example 20) and b (474,475,476M) mix solution were prepared.
- the above fragment a (474), b (474,475,476M) Mix solution was used as a saddle, overlap PCR was performed, and one long fragment (amino acid number 1554 in SEQ ID NO: 7 was substituted with the amino acid at the target position)
- the eye sequence was also made up to the 2663th nucleotide sequence).
- this DNA fragment is referred to as “fragment c (474,475,476M)”.
- the composition of the PCR reaction solution when amplifying fragment c (474,475,476M) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in a cage shape.
- the following DNA was used for the saddle type: Fragments a (474), b (474,475,476M) mix solution 11.
- the PCR reaction conditions are the same as the reaction conditions when fragment c (474,475) was amplified in Example 20.
- the emission spectrum peak of the prepared Q403P / N404G / T405M triple mutant CLuc was 462 nm.
- a gene encoding Q403P, N404G, T405M, and S406L quadruple mutant CLuc (fourth to sixth mutant luciferases) in the amino acid sequence shown in SEQ ID NO: 2 was prepared.
- the method for introducing the mutation is the same as in Example 12.
- a DNA fragment comprising the nucleotide sequence from the 2099th to the 2875th nucleotide in SEQ ID NO: 7 containing the mutation at the 403rd, 404th, 405th and 406th amino acids, This is hereinafter referred to as “fragment b (474,475,476,477)”.
- fragment b 474,475,476,477
- the following oligo DNA primers were used: Q474P / N475G / T476M / S477L-Fw: gtgtccgtcccgtacagctctcccgggatgct catctactggcaagatggtgac (SEQ ID NO: 51) and 3'-UTR ( SEQ ID NO: 19).
- Fragment b (474, The PCR reaction solution composition when amplifying 475,476,477) differs from the PCR reaction solution composition when the fragment b (475X) was amplified in Example 8 only in primers.
- the PCR reaction conditions differed only from the reaction conditions when annealing fragment b (475X) in Example 8 and the annealing temperature, and annealing was performed at 55 ° C.
- fragment c (474), b (474,475,476,477) mix solution as a saddle, overlap PCR was performed, and one long fragment was substituted for the amino acid at the target position (the 1554th force in SEQ ID NO: 7 was 2663 base sequences) were prepared.
- this DNA fragment is referred to as “fragment c (474
- composition of the PCR reaction solution when amplifying fragment c (474,475,476,477) differs from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in the saddle shape. ⁇ type
- Fragments a (474), b (474,475,476,477) mix solution 11.
- the PCR reaction conditions differed only in the reaction conditions and the annealing temperature when amplifying fragment c (475X) in Example 8, and annealing was performed at 61 ° C.
- the emission spectrum peak of the fourth to sixth mutant luciferases was 461 nm.
- a gene encoding A quintet CLuc (4th to 7th mutant luciferase) was prepared.
- the method for introducing the mutation is the same as in Example 12.
- SEQ ID NO: 7 including mutations in the 403rd, 404th, 405th, 406th, and 407th amino acids, the nucleotide sequence from 2099th to 2875th D
- fragment b (474,475,476,477,478)
- fragment b (474,475,476,477,478)
- Q474P / N475G / T476M / S477L / I478A- Fw gtgtccgtcccgtacagctct cccgggatgctcgcctactggcaagatggtgacata (
- the PCR reaction solution composition for amplifying fragment b (474,475,476,477,478) differs from the reaction solution composition for the amplification of fragment b (475X) in Example 8 only in the primer.
- the PCR reaction conditions are the same as the reaction conditions when fragment b (268) was amplified in 16-2 of Example 16.
- Fragment a (474), b (474, 475, 476, 477, 478) Using the mixed solution as a saddle, overlapping PC R was performed, and one long fragment substituted with the amino acid at the target position (position 15 54 in SEQ ID NO: 7) The base sequence up to the 2663th position was also prepared.
- this DNA fragment is referred to as “fragment c (474,475,476,477,478) J”.
- the composition of the PCR reaction solution when amplifying fragment c (474,475,476,477,478) is different from the reaction solution composition when amplifying fragment c (475X) in Example 8 only in the DNA in the saddle shape. .
- the following DNA was used as a cage: fragment a (474), b (474,475,476,477,478) mix solution 11
- the PCR reaction conditions are the same as the reaction conditions when fragment c (249,268) was amplified in Example 16-16-2.
- the emission spectrum peak of the prepared Q403P / N404G / T405M / S406L / I407A five-fold mutant C Luc (fourth to seventh mutant luciferase) was 460 nm.
- the 403rd amino acid was mutated to glutamine proline
- the 404th amino acid was mutated to asparagine glycine
- the 405th amino acid was mutated from threonine to methionine.
- pCLuRA-TDH3 The pCLuRA-TDH3 plasmid in which the 406th amino acid is mutated to serine calucine and the 407th amino acid is mutated from isoleucine to alanine is defined as “pCLuRA-TDH3 [Q403P, N404G, T405M, S406L, I407A]”.
- pCLuRA-TDH3 Q403P, N404G, T405M, S406L, I407A
- fragment a '(238) A DNA fragment containing a mutation at the 167th amino acid and having a base sequence ability up to the first force and 1813th in SEQ ID NO: 7 is hereinafter referred to as "fragment a '(238)".
- the composition of the PCR reaction solution when amplifying fragment a '(238) is different from the reaction solution composition when amplifying fragment a (238) in Example 12 only in the primer.
- the following oligo DNA primers were used: FAR-F (SEQ ID NO: 16) and mut-CLuc-NR2 (SEQ ID NO: 9).
- the PCR reaction conditions differed only from the reaction conditions when the fragment a (475) was amplified in Example 8 and the annealing temperature, and the annealing temperature was 53 ° C.
- fragment comprising the nucleotide sequence from the 1704th position to the 2875th position in SEQ ID NO: 7, including mutations of the 403rd, 404th, 405th, 406th, and 407th amino acids
- fragment 474,475,476,477,478)
- the composition of the PCR reaction solution when amplifying the fragment (474,475,476,477,478) was the same as that of Example 12 and the reaction solution composition when the fragment b (446) was amplified, and only the DNA and primers in the vertical form. Is different.
- the following DNA and oligo DNA primers were used: pCLuRA-TD H3 [Q403P, N404G, T405M, S406L, I407A] (Example 25) 1 ⁇ l (1.45 ng / ⁇ 1), SQ—CLuc— NF2 (SEQ ID NO: 11) and 3′-UTR (SEQ ID NO: 19).
- the PCR reaction conditions are the same as the reaction conditions when the fragment a ′ (238) was amplified.
- fragments a ′(238) and fragments (474,475,476,477,478) were electrophoresed with 1% agarose to confirm that they were DNA fragments of about 1800 bp and about 1200 bp, respectively. These were mixed to prepare fragments a ′ (238), (474,475,476,477,478) mix solution in the same manner as in Example 8.
- fragment c (238,474,475,476,477,478)”.
- the composition of the PCR reaction solution when amplifying fragment c (238,474,475,476,477,478) was the same as that of Example 8 in comparison with the reaction solution composition when amplifying fragment c (475X), The only difference is.
- the following DNA and oligo DNA primers were used: fragment a, (238), b '(474,475,476,477,478) mix solution 1 ⁇ 1, SQ-GPDl-F0 (SEQ ID NO: 41) and mut-CLuc -R ( SEQ ID NO: 13).
- the PCR reaction conditions differed from the reaction conditions for the amplification of fragment c (475X) in Example 8 only in the annealing temperature and extension time.
- the annealing temperature was 51 ° C and the extension time was 2 minutes 30 seconds. It was.
- fragment d (238,474,475,476,477,478) J”.
- the PCR reaction solution composition when amplifying fragment d (238, 474, 475, 476, 477, 478) differs from the reaction solution composition when amplifying fragment d in Example 8 only in the primer.
- the following oligo DNA primers were used in this PCR: SQ-GPD1-R0 (SEQ ID NO: 42) and vec-CLuc-F (SEQ ID NO: 14).
- the PCR reaction conditions are the same as the reaction conditions when fragment d was amplified in Example 8.
- PCR products of fragment c (238,474,475,476,477,478) and fragment d (238,474,475,476,477,478) were 0.7. Electrophoresis with / 0- agarose confirmed that the DNA fragments were about 2200 bp and about 7000 bp, respectively. These were mixed to prepare a mixed solution of fragments c (238,474,475,476,477,478) and d (238,474,475,476,477,478) in the same manner as in Example 8.
- the emission spectrum peak of the prepared T167K / Q403P / N404G / T405M / S406L / I407A six-fold mutant CLuc (third to seventh mutant luciferase) was 461 nm o
- a DNA fragment comprising the first to 2087th nucleotide sequences in SEQ ID NO: 7 containing the mutation at position 21 in SEQ ID NO: 6 and the mutation at position 375 in SEQ ID NO: 2 by PCR did.
- this DNA fragment is referred to as “fragment a (21,446)”.
- the composition of the PCR reaction solution when amplifying fragment a differs from the reaction solution composition when amplifying fragment a (238) in Example 12 only in the DNA and primers.
- the following DNA and oligo DNA primers were used: pCLuRA-TDH3 [a P21L, K375 R] (Example 4) 1 ⁇ l (2.0 ng / ⁇ 1), FAR-F (SEQ ID NO: 16) and SQ-CLuc-F002-rev (SEQ ID NO: 43).
- the PCR reaction conditions are the same as the reaction conditions when fragment a (21) was amplified in 16-3 of Example 16.
- fragment b (474,475,476,477,478).
- the composition of the PCR reaction solution when amplifying fragment b "(474,475,476,477,478) is the same as that of Example 12 and the reaction solution composition of PCR when amplifying fragment a (238) and The following DNA and oligo DNA primers were used: pCLu RA-TDH3 [Q403P, N404G, T405M, S406L, I407A] (Example 25) 1 ⁇ l (1.45 ng / ⁇ 1), SQ—CLuc-F002 (SEQ ID NO: 44) and 3′-UTR (SEQ ID NO: 19)
- the PCR reaction conditions were the same as in Example 16-16-3, in which fragment a (21) was amplified. The reaction conditions are the same.
- fragment c (21,446,474,475,476,477,478)”.
- composition of the PCR reaction solution when amplifying fragment c (21,446,474,475,476,477,478)
- the emission spectrum peak of the prepared K375R / Q403P / N404G / T405M / S406L / I407A hexafold CLuc is 460 nm. fc tsuta.
- a DNA fragment having a nucleotide sequence from the 900th position to the 2087th position in SEQ ID NO: 7 containing a mutation at amino acid numbers 167 and 375 was amplified by PCR.
- this DNA fragment is referred to as “fragment a ′ (238,446)”.
- the composition of the PCR reaction solution when amplifying fragment a '(238,446) is the same as that of the reaction solution composition obtained when amplifying fragment a (238) in Example 12, and only the DNA and primers in the saddle shape. Different. ⁇ The following DNA and oligo DNA primers were used: pCLuRA-TDH3 [T167K, K37 5R] (Example 12) 1 ⁇ l (3.42 ng / ⁇ 1), mut-CLuc-F (SEQ ID NO: 8) and SQ-CLuc-F002-rev (SEQ ID NO: 43). The PCR reaction conditions are the same as the reaction conditions when fragment a (21) was amplified in 16-3 of Example 16.
- Fragment a (21), a '(238,446), b "(474,475,476,477,478) Overlap PCR was performed using the mixed solution as a saddle, and a single long fragment in which the amino acid at the target position was substituted (Consisting of the 461st to 2663th nucleotide sequences in SEQ ID NO: 7) This DNA fragment is hereinafter referred to as “fragment c (21,238,446,474,475,476,477,478)”.
- the composition of the PCR reaction solution when amplifying fragment c differs from the PCR reaction solution composition when amplifying fragment c (238,474,475,476,477,478) in Example 26 only in the DNA in the saddle shape.
- the following DNA was used as the DNA of the type: Fragment a (21), a '(23 8,446), b "(474,475,476,477,478) mix solution 1 1.
- the PCR reaction conditions were the same as in Fragment 16-3 of Example 16. The reaction conditions are the same as when c (21,249,268) was amplified.
- Example 29 Production of mutant CLuc by random mutagenesis to Q403P / N404G / T405M triple mutant CLuc 29-1.
- Introduction of His-tag at C-terminal of Q403P / N404G / T405M triple mutant CLuc In the amino acid sequence shown in SEQ ID NO: 2, triple mutant CLuc (fourth and fifth mutant luciferases of Q403P, N404G and T405M) ), A CLuc- (GS) 3H6 gene in which a His-tag gene was linked downstream of the CLuc gene was prepared.
- the 21st amino acid in the signal sequence of a C Luc shown in SEQ ID NO: 6 was substituted with leucine.
- the following oligo DNA primers were used for PCR to amplify this fragment c '(474,475,476): mut-CLuc-F (SEQ ID NO: 8) and C-trm-r: ctagggtgtctccatgctttatgta (SEQ ID NO: 53) ).
- the PCR reaction solution composition when amplifying fragment c ′ (474,475,476) is V in Example 8, and the reaction solution composition when amplifying fragment c (475X) is the same as that of the DNA and primers in the cage shape. Only is different.
- the following DNAs were used as pegs: pCLuRA-TDH3 [Q403P, N404G, T405 M] (23-2 of Example 23) 1 l (1.82 ng / 1).
- the PCR reaction conditions differed from the reaction conditions when the fragment c (475X) was amplified in Example 8 only in the annealing temperature and the extension time, and the annealing temperature was 57 ° C and the extension time was 2 minutes.
- the following primers were used for PCR in amplifying the fragment d (474,475,476): vec-CLuc-R (SEQ ID NO: 10) and SQ-CLuc-F003: aagctgaacgactctgcaatagtc (SEQ ID NO: 54).
- the PCR reaction solution composition for amplifying fragment d (474,475,476) is different from the reaction solution composition for amplifying fragment d in Example 8 only in the DNA and primers.
- ⁇ -type DNA pCLuRA- TDH3 [a P21L,-(GS) 3H6] (Example 6) 1 ⁇ 1 (1.0 ⁇ ⁇ / ⁇ ⁇ ) 0 PCR reaction conditions were as in Example 8. The reaction conditions are the same as when fragment d was amplified.
- the 403rd amino acid was mutated to glutamine proline
- the 404th amino acid was mutated to asparagine glycine
- the 405th amino acid was also mutated to methionine
- the sequence in the amino acid sequence shown in No. 6 the pCLuRA-TDH3 plasmid in which the 21st amino acid was mutated to proleuca and leucine and the His-tag gene was further introduced downstream of the mutant CLuc gene was designated as ⁇ pCLuRA-TDH3 [a P21L. Q4 03P, N404G, T405M,-(GS) 3H6] ”.
- pCLuRA— TDH3 [a P21L, Q403P, N404G, T405M, — (GS) 3H6] was randomly changed.
- the description will be made using the base number of SEQ ID NO: 23.
- Mutation was introduced by dividing CLuc into the N-terminal side from the 900th position to the 1813th position and the C-terminal side from the 1554th position to the 2699th position in SEQ ID NO: 23 and amplifying them by PCR. The concentration of nucleotides was uneven.
- fragment c (474,475,476) -N”.
- amino acid sequence shown in SEQ ID NO: 2 is amplified by Error Prone PCR, including the 1554th to 2699th nucleotide sequences, which contain mutations at the 403rd, 404th and 405th amino acid positions. did.
- fragment c (474,475,476) -C This DNA fragment is referred to as “fragment c (474,475,476) -C”.
- the composition of Error Prone PCR reaction mixture when amplifying fragment c (474,475,476) -N is as follows: Taq DNA polymerase (Roche) 1 ⁇ 1 (5U / ⁇ 1); pCLuRA— TDH3 [ a P21L,-(G S) 3H6] (Example 6) Plasmid solution (150 ng / 1) 1 1; 10 X PCR buffer w / o Mg 2+ ; for Taq (Roche) 10 1; 10 X dNTP mixture for Error Prone PCR 10 l; 25 mM Magnesium chloride 28 l; 5 mM manganese chloride 2.5 l; mut-CLuc-F (SEQ ID NO: 8) 3 l; mut-CLuc-N R2 (SEQ ID NO: 9) 3 1; sterile water 41.5 ⁇ 1.
- the 10 X dNTP mixture for Error Prone PCR is as follows: lOOmM dCTP 100 ⁇ 1, lOOmM dTTP 100 ⁇ 1, lOOmM dG TP 100 ⁇ 1, lOOmM dATP 100 ⁇ 1, sterile water 760 ⁇ 1.
- the PCR reaction was performed at 94 ° C for 1 minute (denaturation), 45 ° C for 1 minute (annealing), and 72 ° C for 1 minute (extension) in 30 cycles.
- reaction solution composition of Error Prone PCR when amplifying fragment c (474,475,476) -C is the same as the reaction solution composition of Error Prone PCR when fragment c (474,475,476) -N is amplified.
- the amount of DNA, primer and sterilized water is different.
- the amount of DNA, oligo DNA primer, and sterilized water to be used as a cage is as follows: pCLuRA- TDH3 [a P21L, Q403P, N404G, T405M,-(GS) 3H6] (29-1 above) Plasmid solution 0.5 ⁇ l (288 ng / ⁇ 1), mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13), and sterile water 42 1.
- the PCR reaction conditions are the same as those for Error Prone PCR when the fragment c (474,475,476) -N is amplified.
- fragments c (474,475,476) -N and fragment c (474,475,476) -C PCR products with 1% garose an approximately 900 bp fragment c (474,475,476) -N and an approximately 1100 bp fragment were obtained.
- c (474,4 75,476) -C was confirmed. These were each subjected to purification with Sigma GeneElute MINUS EtBr SPIN C OLUMNS, phenol extraction, ethanol precipitation, and then dissolved in 101 1 sterile water (“fragment c (474,475,476) -N solution”, “fragment c ( 474,475,476) -C solution ").
- fragment c (474,475,476) -N and fragment c (474,475,476) -C was amplified by PCR.
- the amplified fragments are defined as “fragment c (474,475,476) -N (2)” and “fragment c (474,475,476) -C (2)”, respectively.
- the composition of the PCR reaction solution for amplifying the fragment c (474,475,476) -N (2) is the same as that of the PCR reaction solution composition obtained when the fragment c (475X) was amplified in Example 8. Only the type of DNA and primers differ. The following DNA and oligo DNA primers were used: Fragment c (474, 475,476) -N solution 1 ⁇ 1, mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-NR2 (SEQ ID NO: 9) .
- the PCR reaction solution composition for amplifying fragment c (474,475,476) -C (2) is the same as the PCR reaction solution composition for amplifying fragment c (474,475,476) -N (2), DNA and primers only Is different.
- the following DNA and oligo DNA primers were used: Fragment c (474,475, 476) -C solution 1 ⁇ 1, mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13) .
- fragment c (474,475,476) -N (2) and fragment c (474,475,476) -C (2) PCR products with 1% agarose an approximately 900 bp fragment c ( 474,475,476) -N (2) and a fragment c (474,475,476) -C (2) of about 11 OObp were confirmed.
- fragment c (474,475,476) - ⁇ (2) solution was dissolved in 20 ⁇ l of sterilized water.
- the PCR reaction solution composition for amplifying fragment d (474,475,476) -N differs from the reaction solution composition for amplification of fragment d in Example 8 only in the DNA and primers.
- the following DNA and oligo DNA primers were used: pCLuRA-TDH3 [a P21L, Q4 03P, N404G, T405M,-(GS) 3H6] (29-1 above) 1 ⁇ l (288 ng / ⁇ 1) Vec-CLuc-R (SEQ ID NO: 10) and SQ-CLuc-NF2 (SEQ ID NO: 11).
- the PCR reaction solution composition used to amplify the fragment d (474,475,476) -C includes the reaction solution composition used when the fragment d (474,475, 476) -N was amplified, and the DNA and primers to be in a cage shape. Only different.
- the following DNA and oligo DNA primers were used: pCLuRA-TDH3 [a P21L,-(GS) 3H6] (Example 6) 1 ⁇ l (150 ng / ⁇ 1), SQ-CLuc-CR1 (sequence) No. 15) and vec-CLu cF (SEQ ID NO: 14).
- the 280th amino acid is mutated to tyrosine strength aspartic acid
- the 372nd amino acid is mutated to arginine leucine
- the 403rd amino acid is mutated to glutamine strength proline.
- the 404th amino acid was mutated to asparagine glycine, the 405th amino acid was mutated from threonine to methionine, and the 21st in the amino acid sequence shown in SEQ ID NO: 6 was mutated to proline leucine, and the CLuc gene
- the pCLuRA-TDH3 plasmid in which the His-tag gene is introduced downstream is defined as “pCLuRA—TDH3 [aP21L, Y280D, R372L, Q403P, N404G, T405M,-(GS) 3H6] j”.
- Example 3O Preparation of mutant CLuc by random mutagenesis to Y280D / R372L / Q403P / N404G / T405M five-fold mutant CLuc pCLuRA—TDH3 [a P21L, Y280D, R372L, Q403P, N404G, T405M, — (GS) 3H6] was randomly mutated.
- the method for introducing the mutation is the same as 29-2 in Example 29.
- SEQ ID NO: 23 This example is described using the base number of SEQ ID NO: 23.
- SEQ ID NO: 23 the nucleotide sequence from the 900th force to the 1717th was amplified by Error Prone PCR. This DNA fragment is referred to as “fragment c (351,443,474,475,476) -N”.
- the amino acid sequence shown in SEQ ID NO: 2 contains mutations at the 280th, 372nd, 403th, 404th and 405th amino acid positions, from 1554th to 2699th.
- the nucleotide sequence up to was amplified by Error Prone PCR. This DNA fragment is referred to as “fragment c (351,443,474,475,476) -C”.
- pCLuRA- TDH3 [a P21L, Y280D, R372L, Q403P, N404G, T405M,-(GS) 3H6] (Example 29-29-2) 0.5 ⁇ l (329ng / ⁇ 1), mut-CLuc-F (SEQ ID NO: 8) and ⁇ 340—rev: gtacggctcgagaagaccttt (
- the reaction solution composition of Error Prone PCR when amplifying fragment c (351,443,474,475,476) -C is different from Error Prone PCR when amplifying fragment c (351,443,474,475,476) -N only in primers.
- the oligo DNA primers used were: mut-CLuc-CFl (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13).
- Fragment c (351,443,474,475,476) -N solution and fragment c (351,443,474,475,476) -C solution were prepared in the same manner as Example 29-2.
- fragment c (351,443,474,475,476) -N and fragment c (351,443,474,475,476) -C was amplified by PCR.
- the amplified fragments are referred to as “fragment c (351,443,474,475,476) -N (2) j” and “fragment c (351,443,474,475,476) -C (2)”, respectively.
- the PCR reaction solution composition for amplifying fragment c (351,443,474,475,476) -C (2) was the same as that for fragment c (474,475,476) -C (2) in Example 29-2. The only difference is the composition of the reaction solution and the DNA of the cage type. The following DNA was used for the cage type: Fragment c (351,443,474,475,476) -C solution.
- fragment d (351,443,474,475,476) -N”.
- the composition of the PCR reaction solution when amplifying fragment d (351,443,474,475,476) -N is the same as that in Example 8; the PCR reaction solution composition when fragment d was amplified; Only the difference is.
- the following DNA and oligo DNA primers were used: pCLuRA-TD H3 [a P21L, Y280D, R372L, Q403P, N404G, T405M,-(GS) 3H6], vec-CLuc-R (SEQ ID NO: 10 ) And SQ-CLuc-NF2 (SEQ ID NO: 11).
- the PCR reaction conditions are the same as the PCR reaction conditions when fragment d was amplified in Example 8.
- fragment c (351,443,474,475,476) -N (2) solution 10 ⁇ 1 and fragment d (351,443,474,475,476) -N solution 5 ⁇ 1 were mixed, and fragment c (351,443,474,475,476) -N (2), d (351,443,474,475,476) -A mix solution was prepared.
- fragment c (351,443,474,475,476) -C (2) solution 10 1 and the fragment d (4 74,475,476) -C solution (29-2 of Example 29) 51 are mixed to obtain the fragment c (351,443,474,475,476) -C (2 ), d (474,475,476) -C mix solution was prepared.
- Example 31 Preparation of mutant CLuc by random mutagenesis to L191Q / Y280D / R372L / Q403P / N404G / T405M hexafold CLuc pCLuRA-TDH3 [a P21L, L191Q, Y280D, R372L, Q403P, N404G, T405M- (GS) 3H6] was randomly mutated. Mutation introduction method is the same as 29-2 in Example 29
- the amount of DN A and sterilized water to be used was the following: pCLuRA- TDH3 [a P21L, L191Q, Y280D, R372L, Q 403P, N404G, T405M- (GS) 3H6] (Example 30) 0.5 ⁇ l (298 ng / ⁇ 1) and sterile water 42 1.
- the reaction conditions of Error Prone PCR when amplifying fragment c (262,351,443,474,475,476) -N were the same as those when fragment c (474,475,476) -N and fragment c (474,475,476) -C were amplified in Example 29-29-2. The conditions are the same.
- fragment c (262,351,443,474,475,476) -N (2)”.
- the PCR reaction solution composition when amplifying fragment c (262,351,443,474,475,476) -N (2) is the same as the reaction solution composition obtained when amplifying fragment c (475X) in Example 8, and Only the DNA and primers that differ are different.
- the following DNA and oligo DNA primers were used as fragments: fragment c (262,351,443,474,475,476) -N solution, mut-CLuc-F (SEQ ID NO: 8) and SQ-CLuc-CR1 (SEQ ID NO: 15).
- the composition of the reaction solution when amplifying the fragment d (262,351,443,474,475,476) -C is the same as the composition of the reaction solution when the fragment d (474,475,476) -C is amplified in 29-2 of Example 29. Only the DNA is different.
- the following DNAs were used for the DNA: pCLuRA- TDH3 [a P21L, L191Q, Y280 D, R372L, Q403P, N404G, T405M- (GS) 3H6] o Also amplified fragment d (262,351,443,474,475,476) -C
- the PCR reaction conditions for this are the same as the reaction conditions for the amplification of fragment d in Example 8.
- fragment c (351,443,474,475,476) -N (2) solution (Example 30) 10 / z 1 and the fragment d (262,351,443,474,475,476) -C solution 5 / z 1 were mixed and the fragment c (351, 443,474,475,476) -N (2), d (262,351,443,474,475,476) mix solution was prepared.
- the emission spectrum peak of the selected L191Q / Q235R / Y280D / R372L / Q403P / N404G / T405M heptad mutant CLuc was 466 nm.
- the emission spectrum peak of M178R / L191Q / Y280D / R372L / Q403P / N404G / T405M hepta mutant CLuc (9th mutant luciferase) was 435 nm.
- the difference between the peak wavelengths of these two variants, CLuc, is 31 and can be sufficiently separated by optical filter and program analysis. Two mutant luciferases with different emission colors were obtained.
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WO2008148519A3 (en) * | 2007-06-04 | 2009-05-22 | Lonza Biologics Plc | Mammalian expression vector with a highly efficient secretory signal sequence |
JP2009207447A (ja) * | 2008-03-05 | 2009-09-17 | National Institute Of Advanced Industrial & Technology | 変異型ルシフェラーゼ |
JP2012249619A (ja) * | 2011-06-07 | 2012-12-20 | National Institute Of Advanced Industrial Science & Technology | 海洋プランクトン由来発光タンパク質 |
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US10385319B2 (en) | 2016-09-08 | 2019-08-20 | The Governement of the United States of America, as represented by the Secretary of Homeland Security | Modified foot-and-mouth disease virus 3C proteases, compositions and methods thereof |
US10435695B2 (en) * | 2016-09-08 | 2019-10-08 | The Government of the United States of America, as represented by the Secretary of Homeland Security | Fusion protein comprising Gaussia luciferase, translation interrupter sequence, and interferon amino acid sequences |
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US7871803B2 (en) * | 2004-12-09 | 2011-01-18 | Nec Soft, Ltd. | Gene encoding novel luciferase |
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Also Published As
Publication number | Publication date |
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US20090263880A1 (en) | 2009-10-22 |
JP5224457B2 (ja) | 2013-07-03 |
GB2452457A (en) | 2009-03-04 |
GB0823611D0 (en) | 2009-02-04 |
US8147842B2 (en) | 2012-04-03 |
JPWO2007144990A1 (ja) | 2009-10-29 |
GB2452457B (en) | 2011-05-25 |
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