# Patent

## Title
Mutant luciferase

## Abstract

An object of the present invention is to provide a mutant luciferase having luciferase activity with an altered emission spectrum. A specific amino acid residue(s) is substituted in a luciferase derived from Cypridina noctiluca and then the resulting mutant luciferase having luciferase activity with an emission spectrum differing from that of the wild-type luciferase is screened for.

## Background
TECHNICAL FIELD
The present invention relates to a mutant luciferase having luciferase activity with an altered emission spectrum, for example.
BACKGROUND OF THE INVENTION
Reporter assay is a means for quantifying the transcriptional activity of a transcriptional regulatory sequence. Reporter assay is carried out by ligating a gene (hereinafter, referred to as “reporter gene”) encoding a reporter protein under the control of a transcriptional regulatory sequence (e.g., promoter and enhancer) to be examined, introducing the resultant into a host cell, and then causing the expression of the protein. At this time, the transcriptional activity of a promoter is positively correlated with the amount of reporter protein generated by transcription and translation, for example. Hence, through quantification of the amount of reporter protein, the degree (high or low) of the relative transcriptional activity of a promoter can be evaluated.
Reporter assay can be carried out using various proteins as reporter proteins. For example, when a fluorescent protein is used as reporter protein, the thus expressed fluorescent protein is irradiated with excitation light and then the thus generated fluorescence intensity is measured, so that the relative amount of the reporter protein can be quantified (this method is referred to as a fluorescence method).
Furthermore, for example, reporter assay can be carried out using an enzyme such as β-galactosidase or alkaline phosphatase as a reporter protein. When an enzyme is used as a reporter protein, the relative amount of the reporter protein can be quantified by colorimetry with the use of a substrate that is degraded by the action of the enzyme so as to generate a color substance (this method is referred to as calorimetric method). Another method involves the use of a substrate that causes the generation of a luminescent substance instead of a substrate that causes the generation of a color substance. In this case, the relative amount of reporter protein can be quantified through measurement of the amount of luminescence (this method is referred to as a luminescent method).
Such a luminescent method has the following excellent characteristics. First, the method does not require any excitation light unlike a fluorescence method, so that the background is small and a high ratio of signal to noise can be obtained. Furthermore, the method has high sensitivity, by which a broad dynamic range can be obtained. Moreover, the method is excellent in terms of quantitative capability.
An example of an enzyme reaction system that is generally used in the luminescent method is a luciferase/luciferin reaction system.
Various types of luciferase are known, and they differ significantly from each other in terms of primary structure. For example, there are various luciferases derived from various organisms, including firefly and Renilla. 
Meanwhile, various types of luciferins are present as substrates, which differ greatly from each other in terms of chemical structure.
Types of luciferin that each luciferase recognizes as a substrate are limited to some extent. A technique generally referred to as a dual reporter assay involves adding a luciferin derived from firefly (hereinafter, referred to as “firefly luciferin”) and a luciferin derived from Renilla (hereinafter, referred to as “Renilla luciferin”) (coelenterazine) successively to a sample solution containing a mixture of a luciferase derived from firefly (hereinafter, referred to as “firefly luciferase”) and a luciferase derived from Renilla (hereinafter, referred to as “Renilla luciferase”) and then separately measuring the activity of the firefly luciferase and the activity of the Renilla luciferase.
Sea-firefly includes species such as Vargula hilgendorfii and Cypridina noctiluca. In such species of sea-firefly, luciferase is released ex vivo (specifically, in sea water) and then luciferin reacts with oxygen in sea water because of the catalytic action of the luciferase so as to produce luminescence.
Genes encoding a luciferase derived from Vargula hilgendorfii (hereinafter, referred to as “VLuc”) and a luciferase derived from Cypridina noctiluca (hereinafter, referred to as “CLuc”), respectively, have been cloned (Thompson, E. M., Nagata S., Tsuji F. I., “Proceedings of the National Academy of Sciences of the United States of America,” 1989, Vol. 86, p. 6567-6571; and Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., Ohmiya, Y., “Bioscience and Biotechnology and Biochemistry,” 2004, Vol. 68, p. 565-570). Both VLuc and CLuc are expressed in cultured cells and can be caused to be secreted extracellularly (JP Patent Publication (Kokai) No. 3-30678 A (1991) and International Publication No. 2006/132350 Pamphlet). Specifically, VLuc and CLuc are secretory luciferases. Therefore, such a gene encoding the luciferase (hereinafter, referred to as a “luciferase gene”) is used as a reporter gene, and the transcriptional activity of a transcriptional regulatory sequence such as a promoter can be measured without disrupting cells (International Patent Publication No. 2006/132350 Pamphlet).
In the case of secretory luciferases, a culture solution containing a secretory luciferase can be directly used as a solution to be tested. Hence, secretory luciferases are appropriate for construction of, namely, a high-throughput reporter assay system for treatment of many samples. On the other hand, in the case of non-secretory luciferases, collection of cells by centrifugation and disruption (or enhancement of cell permeabilization) of cells by ultrasonication, treatment with a surfactant, treatment with an organic solvent, or the like are essential. These procedures are inappropriate for treatment of numerous samples. Furthermore, in the case of secretory luciferases, a sample for measurement can be obtained by collecting a portion of a culture solution without disrupting cells. Thus, sampling can be carried out consecutively for the same cells. On the other hand, in the case of non-secretory luciferases, cells are always damaged by cell disruption or the like. Hence, consecutive sampling with the use of the same cells is impossible and as many different cells should be prepared as the number of measurement points.
It has been reported that the above CLuc is secreted in a culture solution at a level 320 times greater than VLuc, when expressed in NIH3T3 cells, and 410 times greater than VLuc, when expressed in HeLaS3 cells (Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., Ohmiya, Y., “Bioscience and Biotechnology and Biochemistry,” 2004, Vol. 68, p. 565-570). Therefore, compared with VLuc, CLuc is appropriate for use in a high-sensitivity, high-throughput reporter assay system using cultured cells as hosts.
Furthermore, a secretory high-throughput reporter assay system using a budding yeast Saccharomyces cerevisiae into which a CLuc gene has been introduced has also been conceived (International Publication No. 2006/132350 Pamphlet).
The luminescence mechanism of a luciferase/luciferin reaction system is generally considered to be as follows. First, luciferin is oxidized by catalytic action of luciferase into oxyluciferin in its excited state. Subsequently, oxyluciferin in its excited state immediately returns to the ground state, during which the oxyluciferin releases energy in the form of light (produces luminescence). The amount of luminescence produced per unit of time is thought to be proportional to the amount of luciferase existing in the system. Thus, the relative amount of luciferase can be quantified based on the luminescence.
With the above luminescence mechanism, luminescence can be obtained in accordance with the difference in energy level between the excited state and the ground state of oxyluciferin. A change in energy level difference appears as an emission spectrum change. Specifically, when the energy level in the excited state changes for some reason upon production of luminescence, luminescence with a color differing from that of a general case is obtained. This effect is known to take place due to a significant difference or a local difference in terms of the primary structure of luciferase (Viviani, V., Uchida, A., Suenaga, N., Ryufuku, M., Ohmiya, Y., “Biochemistry and Biophysics Research Communication,” 2001, Vol. 280, p. 1286-1291).
Meanwhile, there are at least two methods (referred to as “multi-reporter assay”) for simultaneously carrying out reporter assays of 2 or more types of promoter activity using a luciferase gene as a reporter gene, as follows.
A first method involves the use of a plurality of different chemical species of luciferins and luciferases having substrate specificity for each luciferin. In the case of this method, no reaction takes place with combinations other than the combination of a luciferase and a luciferin that form a pair, because of differences in substrate specificity. Furthermore, appropriate conditions (e.g., compositions of reaction solutions and hydrogen ion concentrations) differ depending on the reaction of each luciferase/luciferin reaction system. In the case of this method, reaction conditions should be varied depending on each luciferase/luciferin reaction, for one specimen, and the reactions should be carried out in order or in parallel. In accordance therewith, multiple luminescence measurements should be carried out for one specimen by employing different conditions appropriate for each luciferase/luciferin reaction. As described above, this method is problematic in terms of its complicated measurement procedures.
A second method involves the use of luciferins of the same chemical species as substrates. In this case, multiple types of luciferase whose substrates are luciferins of the same chemical species are used as reporter proteins. The amino acid sequences of these luciferases partially differ from each other, and they are characterized in that different emission spectra are generated from luciferases. The luminescence intensity originating from each luciferase should be determined and quantified based on differences in spectrum.
A multi-reporter assay using the above second method has the advantage of being simple because only a single type of substrate is used, and because the luminous reaction and the measurement can each be completed at one time.
In the case of luminescence simultaneously produced from luciferases with different luminescent colors, the spectra thereof may overlap. However, even under such circumstances, a method for estimating the luminescence intensity originating from each luciferase has been conceived (JP Patent Publication (Kokai) No. 2004-333457 A).
An example of a multi-reporter assay using the principle of the above second method is a method that involves the use of a luciferase gene derived from a luminescent beetle and a mutant gene thereof (Yoshihiro Nakajima and Yoshihiro Ohmiya, “Biotechnology Journal,” 2006, Vol. 6, No. 2, p. 230-232). However, such a luciferase derived from a luminescent beetle is non-secretory. Therefore, the luciferase is inappropriate for use in high-throughput reporter assays for the reasons as described above.
As described above, no high-throughput multi-reporter assay using the principle of the above second method is currently known. Furthermore, concerning sea-firefly luciferase, the presence of any mutant luciferase that alters luminescent color is unknown.
Meanwhile, a phenomenon referred to as BRET (Bioluminescence resonance energy transfer) is used as a method for detecting the structural changes of proteins at the biochemical level or the cellular level, for example (Otsuji, T., Okuda-Ashitaka, E., Kojima, S., Akiyama, H., Ito, S., Ohmiya, Y., “Analytical Biochemistry,” 2004, Vol. 329, p. 230-237).
In BRET, a luminous object and a fluorescing object form a pair. As a luminous object, a bioluminescent substance such as luciferase, luciferin, or the like is used. On the other hand, as a fluorescing object, for example, a chemical substance producing fluorescence or a fluorescent protein such as a green fluorescent protein (GFP) is used. When a luminous object and a fluorescing object are located at positions that enable (in terms of distance) topological energy transfer, the luminous object is excited and the energy released when it returns to the ground state is transferred to the fluorescing object. Subsequently the fluorescing object is excited and it emits light when it returns to the ground state. Each fluorescing object has its unique excitation spectrum and excitation efficiency is known to depend on the emission spectrum of the luminous object and the excitation spectrum of the fluorescing object. A luminous object emitting light at a wavelength that efficiently excites a fluorescing object is most preferable for composing an efficient BRET pair.
Hence, luciferase to be used in BRET analysis using luciferase and luciferin as luminous objects preferably emits light at a wavelength that efficiently excites the fluorescing object, with reference to the excitation spectrum of a fluorescing object to be used in the form of a pair. Therefore, the presence of mutant luciferases emitting light at different wavelengths makes it possible to form BRET pairs appropriate for various fluorescing objects.
DISCLOSURE OF THE INVENTION
Object to be Achieved by the Invention
In view of the above circumstances, an object of the present invention is to provide a mutant luciferase having luciferase activity with an altered emission spectrum.
Means to Achieve the Object
As a result of intensive studies to achieve the above object, it has been found that a specific amino acid residue can be substituted in luciferase (CLuc) derived from Cypridina noctiluca, so that a mutant luciferase having luciferase activity that exhibits an emission spectrum differing from that of a wild-type luciferase can be obtained. Thus, the present invention has been completed.
The present invention encompasses the following (1) to (34).
(1) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of lysine at position 375 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 375 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of lysine at position 375 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 375 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more.
(2) The mutant luciferase according to (1), wherein lysine at position 375 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine.

(3) The mutant luciferase according to (1), wherein the emission spectral peak ranges from 457 nm to 490 nm.

(4) A mutant luciferase consisting of any one of the following proteins (a) to (d):

(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of methionine at position 178 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 178 of such amino acid and having luciferase activity with an emission spectral peak of 449 nm or less;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of methionine at position 178 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 178 of such amino acid and having luciferase activity with an emission spectral peak of 449 nm or less.
(5) The mutant luciferase according to (4), which has the substitution of methionine at position 178 with lysine.
(6) The mutant luciferase according to (4), wherein the emission spectral peak ranges from 420 nm to 449 nm.
(7) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of threonine at position 167 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 167 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of threonine at position 167 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 167 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more.
(8) The mutant luciferase according to (7), which has the substitution of threonine at position 167 with lysine.
(9) The mutant luciferase according to (7), wherein the emission spectral peak ranges from 458 nm to 490 nm.
(10) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of asparagine at position 404 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 404 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of asparagine at position 404 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 404 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more.
(11) The mutant luciferase according to (10), which has the substitution of asparagine at position 404 with glycine or serine.
(12) The mutant luciferase according to (10), wherein the emission spectral peak ranges from 458 nm to 490 nm.
(13) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of threonine at position 405 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 405 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of threonine at position 405 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 405 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more.
(14) The mutant luciferase according to (13), which has the substitution of threonine at position 405 with isoleucine or methionine.
(15) The mutant luciferase according to (13), wherein the emission spectral peak ranges from 457 nm to 490 nm.
(16) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of serine at position 406 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 406 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of serine at position 406 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 406 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more.
(17) The mutant luciferase according to (16), which has the substitution of serine at position 406 with leucine.
(18) The mutant luciferase according to (16), wherein the emission spectral peak ranges from 460 nm to 490 nm.
(19) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of isoleucine at position 407 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 407 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of isoleucine at position 407 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 407 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more.
(20) The mutant luciferase according to (19), which has the substitution of isoleucine at position 407 with alanine.
(21) The mutant luciferase according to (19), wherein the emission spectral peak ranges from 460 nm to 490 nm.
(22) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to amino acid sequence shown in SEQ ID NO: 2, substitutions of leucine at position 191, glutamine at position 235, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 466 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and substitutions of leucine at position 191, glutamine at position 235, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 466 nm or more.
(23) The mutant luciferase according to (22), containing the following amino acid substitutions (A) to (G):
(A) a substitution of leucine at position 191 with glutamine;
(B) a substitution of glutamine at position 235 with arginine;
(C) a substitution of tyrosine at position 280 with aspartic acid;
(D) a substitution of arginine at position 372 with leucine;
(E) a substitution of glutamine at position 403 with proline;
(F) a substitution of asparagine at position 404 with glycine; and
(G) a substitution of threonine at position 405 with methionine.
(24) The mutant luciferase according to (22), wherein the emission spectral peak ranges from 466 nm to 490 nm.
(25) A mutant luciferase consisting of any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, substitutions of methionine at position 178, leucine at position 191, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 435 nm or less;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and substitutions of methionine at position 178, leucine at position 191, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 435 nm or less.
(26) The mutant luciferase according to (25), containing the following amino acid substitutions (A) to (G):
(A) a substitution of methionine at position 178 with arginine;
(B) a substitution of leucine at position 191 with glutamine;
(C) a substitution of tyrosine at position 280 with aspartic acid;
(D) a substitution of arginine at position 372 with leucine;
(E) a substitution of glutamine at position 403 with proline;
(F) a substitution of asparagine at position 404 with glycine; and
(G) a substitution of threonine at position 405 with methionine.
(27) The mutant luciferase according to (25), wherein the emission spectral peak ranges from 420 nm to 435 nm.
(28) A fusion protein, wherein a foreign protein or peptide is linked to the mutant luciferase according to any one of (1) to (27).
(29) A gene encoding the mutant luciferase according to any one of (1) to (27) or the fusion protein according to (28).
(30) A recombinant vector containing the gene according to (29).
(31) A transformant having the recombinant vector according to (30).
(32) The transformant according to (31), wherein two or more genes selected from the group consisting of the gene according to (29) and the genes encoding luciferases or fusion protein consisting of the following proteins (a) to (c) are each placed under the control of different promoters:
(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18; and
(c) a fusion protein in which a foreign protein or peptide is linked to the protein (a) or (b).
(33) A method for evaluating the transcriptional activity of a promoter, comprising a step of causing a culture or a culture supernatant of the transformant according to (32) to come into contact with a luciferin or a derivative thereof and a step of measuring the luminescence intensity of the emission spectrum based on the activity of each luciferase, wherein the transcriptional activity of 2 or more promoters is evaluated.

(34) A method for emitting light or a method for releasing energy, comprising a step of causing the mutant luciferase according to any one of (1) to (27) or the fusion protein according to (28) to come into contact with a luciferin or a derivative thereof and a step of causing an oxyluciferin or a derivative thereof in its excited state to act on a chemical substance, wherein light emission or energy release is caused based on excitation of the chemical substance.

Effects of the Invention
According to the present invention, a mutant luciferase having luciferase activity resulting in an emission spectrum differing from that of a wild-type luciferase can be provided. Furthermore, with the use of the mutant luciferase according to the present invention, a simple and highly sensitive multi-reporter assay system can be provided. Moreover, upon BRET analysis, the mutant luciferase according to the present invention can be an excellent energy donor.
The present specification includes part or all of the contents as disclosed in the specification and/or drawing of Japanese Patent Application No. 2006-162662, which is a priority document of the present application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the relative luminescence intensity (or relative light unit) to wavelength of each luciferase.



PREFERRED EMBODIMENTS OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
A 1st mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 1st mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of lysine at position 375 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 375 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of lysine at position 375 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 375 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more.
The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is a luciferase (CLuc) derived from Cypridina noctiluca. Furthermore, the nucleotide sequence shown in SEQ ID NO: 1 is a gene (cDNA) encoding CLuc.
The mutant luciferase according to (a) above among the 1st mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of lysine at position 375 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 457 nm or more and particularly the emission spectral peak ranging from 457 nm to 490 nm (e.g., 457 nm to 463 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than lysine.
Meanwhile, the mutant luciferase according to (b) above among the 1st mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 375 of such amino acid and has luciferase activity with an emission spectral peak of 457 nm or more. Examples of the positions other than position 375 of the amino acid include threonine at position 167, glutamine at position 403, asparagine at position 404, threonine at position 405, serine at position 406, and isoleucine at position 407.
Furthermore, the mutant luciferase according to (c) above among the 1st mutant luciferases is a mature protein in which a secretory signal peptide (consisting of an amino acid sequence ranging from amino acids at positions 1 to 18 of the amino acid sequence shown in SEQ ID NO: 2) of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a). In general, secretory proteins including CLuc are synthesized in the forms of precursors each having a secretory signal peptide on the N-terminus. Such a precursor is cleaved with a signal peptidase during a transmembrane process to be a mature protein. In the present invention, the term “mature protein” refers to a protein that is secreted outside the cell membranes or cell walls.
Furthermore, the mutant luciferase according to (d) above among the 1st mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 2nd mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 2nd mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of methionine at position 178 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 178 of such amino acid and having luciferase activity with an emission spectral peak of 449 nm or less;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of methionine at position 178 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 178 of such amino acid and having luciferase activity with an emission spectral peak of 449 nm or less.
The mutant luciferase according to (a) above among the 2nd mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of methionine at position 178 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 449 nm or less and particularly the emission spectral peak ranging from 420 nm to 449 nm (e.g., 447 nm to 449 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than methionine and is particularly desirably lysine.
Meanwhile, the mutant luciferase according to (b) above among the 2nd mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 178 of such amino acid and has luciferase activity with an emission spectral peak of 449 nm or less. An example of the positions other than position 178 of the amino acid is leucine at position 197.
Furthermore, the mutant luciferase according to (c) above among the 2nd mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 2nd mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 3rd mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 3rd mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of threonine at position 167 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 167 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of threonine at position 167 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 167 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more.
The mutant luciferase according to (a) above among the 3rd mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of threonine at position 167 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 458 nm or more and particularly the emission spectral peak ranging from 458 nm to 490 nm (e.g., 458 nm to 460 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than threonine and examples of which include isoleucine, leucine, and lysine and is particularly desirably isoleucine or lysine.
Meanwhile, the mutant luciferase according to (b) above among the 3rd mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 167 of the amino acid and has luciferase activity with an emission spectral peak of 458 nm or more. Examples of the positions other than position 167 of the amino acid include lysine at position 375, glutamine at position 403, asparagine at position 404, threonine at position 405, serine at position 406, and isoleucine at position 407.
Furthermore, the mutant luciferase according to (c) above among the 3rd mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 3rd mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 4 h mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 4th mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of asparagine at position 404 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 404 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of asparagine at position 404 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 404 of such amino acid and having luciferase activity with an emission spectral peak of 458 nm or more.
The mutant luciferase according to (a) above among the 4th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of asparagine at position 404 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 458 nm or more and particularly the emission spectral peak ranging from 458 nm to 490 nm (e.g., 458 nm to 460 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than asparagine. Examples of such amino acid include glycine, alanine, serine, and threonine and a particularly desirable example thereof is glycine or serine.
Meanwhile, the mutant luciferase according to (b) above among the 4th mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 404 of the above amino acid and has luciferase activity with an emission spectral peak of 458 nm or more. Examples of the positions other than position 404 of the amino acid include lysine at position 38, serine at position 45, valine at position 75, arginine at position 79, arginine at position 87, aspartic acid at position 112, lysine at position 126, threonine at position 167, glutamic acid at position 170, leucine at position 191, methionine at position 223, glutamine at position 235, valine at position 258, isoleucine at position 276, tyrosine at position 280, methionine at position 291, threonine at position 313, arginine at position 372, lysine at position 375, glutamine at position 403, threonine at position 405, serine at position 406, isoleucine at position 407, and glutamic acid at position 479.
Furthermore, the mutant luciferase according to (c) above among the 4th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 4th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 5th mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 5th mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of threonine at position 405 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 405 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of threonine at position 405 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 405 of such amino acid and having luciferase activity with an emission spectral peak of 457 nm or more.
The mutant luciferase according to (a) above among the 5th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of threonine at position 405 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 457 nm or more and particularly the emission spectral peak ranging from 457 nm to 490 nm (e.g., 457 nm to 460 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than threonine. Examples of such amino acid include isoleucine, methionine, and leucine and a particularly desirable example thereof is isoleucine or methionine.
Meanwhile, the mutant luciferase according to (b) above among the 5th mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 405 of the above amino acid and has luciferase activity with an emission spectral peak of 457 nm or more. Examples of the positions other than position 405 of the amino acid include lysine at position 38, serine at position 45, valine at position 75, arginine at position 79, arginine at position 87, aspartic acid at position 112, lysine at position 126, threonine at position 167, glutamic acid at position 170, leucine at position 191, methionine at position 223, glutamine at position 235, valine at position 258, isoleucine at position 276, tyrosine at position 280, methionine at position 291, threonine at position 313, arginine at position 372, lysine at position 375, glutamine at position 403, asparagine at position 404, serine at position 406, isoleucine at position 407, and glutamic acid at position 479.
Furthermore, the mutant luciferase according to (c) above among the 5th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 5th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 6th mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 6th mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of serine at position 406 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 406 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of serine at position 406 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 406 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more.
The mutant luciferase according to (a) above among the 6th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of serine at position 406 with another amino acid. This protein exerts luciferase activity similarly to that of CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 460 nm or more and particularly the emission spectral peak ranging from 460 nm to 490 nm (e.g., 460 nm to 462 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than serine. Examples of such amino acid include leucine and isoleucine and a particularly desirable example thereof is leucine.
Meanwhile, the mutant luciferase according to (b) above among the 6th mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 406 of the above amino acid and has luciferase activity with an emission spectral peak of 460 nm or more. Examples of the positions other than position 406 of the amino acid include threonine at position 167, lysine at position 375, glutamine at position 403, asparagine at position 404, threonine at position 405, and isoleucine at position 407.
Furthermore, the mutant luciferase according to (c) above among the 6th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 6th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
Meanwhile, a 7th mutant luciferase according to the present invention is any one of the following proteins (a) to (d) (hereinafter, referred to as “the 7th mutant luciferase(s)”):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a substitution of isoleucine at position 407 with another amino acid;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 407 of the above amino acid and having luciferase activity with an emission spectral peak of 460 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and a substitution of isoleucine at position 407 with another amino acid; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than position 407 of such amino acid and having luciferase activity with an emission spectral peak of 460 nm or more.
The mutant luciferase according to (a) above among the 7th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, a substitution of isoleucine at position 407 with another amino acid. This protein exerts luciferase activity similarly to CLuc. However, because of the amino acid substitution, the protein is characterized by the emission spectral peak of 460 nm or more and particularly the emission spectral peak ranging from 460 nm to 490 nm (e.g., 460 nm to 462 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, “another amino acid” may be any amino acid other than isoleucine. Examples of such amino acid include glycine and alanine and a particularly desirable example thereof is alanine.
Meanwhile, the mutant luciferase according to (b) above among the 7th mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than position 407 of the above amino acid and has luciferase activity with an emission spectral peak of 460 nm or more. Examples of the positions other than position 407 of the amino acid include threonine at position 167, lysine at position 375, glutamine at position 403, asparagine at position 404, threonine at position 405, and serine at position 406.
Furthermore, the mutant luciferase according to (c) above among the 7th mutant luciferases is a mature protein from which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 7th mutant luciferases is a mature protein from which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
The mutant luciferases according to the present invention also includes a protein containing multiple amino acid substitutions that form a combination of any two or more (e.g., 2 to 10, preferably 2 to 8, and particularly preferably 2 to 6) of each predetermined amino acid substitution in the 1st and the 3rd to the 7th mutant luciferases above and amino acid substitutions at the other positions in the amino acid sequence shown in SEQ ID NO: 2, and having luciferase activity with an emission spectral peak of 458 nm or more and particularly an emission spectral peak ranging from 458 nm to 490 nm (e.g., 458 nm to 475 nm). An example of such mutant luciferase containing such multiple amino acid substitutions is an 8th mutant luciferase (hereinafter, referred to as “the 8th mutant luciferase(s)”) represented by any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to amino acid sequence shown in SEQ ID NO: 2, a substitution of leucine at position 191, glutamine at position 235, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 466 nm or more;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and substitutions of leucine at position 191, glutamine at position 235, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 466 nm or more.
The mutant luciferase according to (a) above among the 8th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, in addition to substitutions of asparagine at position 404 corresponding to the position of an amino acid substitution in the 4th mutant luciferases and threonine at position 405 corresponding to the position of an amino acid substitution in the 5th mutant luciferases, substitutions of leucine at position 191, glutamine at position 235, tyrosine at position 280, arginine at position 372, and glutamine at position 403 with other amino acids. This protein exerts luciferase activity similarly to CLuc. However, because of the amino acid substitutions, the protein is characterized by an emission spectral peak of 466 nm or more and particularly an emission spectral peak ranging from 466 nm to 490 nm (e.g., 466 nm to 475 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, examples of the amino acid substitution at each amino acid position include a combination of the following (A) to (G):
(A) a substitution of leucine at position 191 with glutamine;
(B) a substitution of glutamine at position 235 with arginine;
(C) a substitution of tyrosine at position 280 with aspartic acid;
(D) a substitution of arginine at position 372 with leucine;
(E) a substitution of glutamine at position 403 with proline;
(F) a substitution of asparagine at position 404 with glycine; and
(G) a substitution of threonine at position 405 with methionine.
Meanwhile, the mutant luciferase according to (b) above among the 8th mutant luciferases consists of an amino acid sequence that has, with respect to the mutant luciferase according to (a), a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than the predetermined positions of the above amino acids and has luciferase activity with an emission spectral peak of 466 nm or more. Examples of the positions other than the predetermined positions of the above amino acids include aspartic acid at position 112, methionine at position 291, and threonine at position 313.
Furthermore, the mutant luciferase according to (c) above among the 8th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 8th mutant luciferases is a mature protein in which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
The mutant luciferases according to the present invention also include a protein containing multiple amino acid substitutions that form a combination of any two or more (e.g., 2 to 10, preferably 2 to 8, and particularly preferably 2 to 6) of the amino acid substitution in the 2nd mutant luciferases and amino acid substitutions at the other positions in the amino acid sequence shown in SEQ ID NO: 2, and having luciferase activity with an emission spectral peak of 449 nm or less and particularly an emission spectral peak ranging from 420 nm to 449 nm (e.g., 425 nm to 449 nm). An example of such mutant luciferase containing such multiple amino acid substitutions is a 9th mutant luciferase (hereinafter, referred to as “the 9th mutant luciferase(s)”) represented by any one of the following proteins (a) to (d):
(a) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, substitutions of methionine at position 178, leucine at position 191, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids;
(b) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (a) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 435 nm or less;
(c) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence shown in SEQ ID NO: 2, a deletion of amino acids at positions 1 to 18 and substitutions of methionine at position 178, leucine at position 191, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids; and
(d) a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of (c) above, a deletion, a substitution, or an addition of one or several amino acids at positions other than the positions of such amino acids and having luciferase activity with an emission spectral peak of 435 nm or less.
The mutant luciferase according to (a) above among the 9th mutant luciferases is a protein consisting of an amino acid sequence that has, with respect to the amino acid sequence of CLuc, in addition to the substitution of methionine at position 178 corresponding to the position of an amino acid substitution in the 2nd mutant luciferases, substitutions of leucine at position 191, tyrosine at position 280, arginine at position 372, glutamine at position 403, asparagine at position 404, and threonine at position 405 with other amino acids. This protein exerts luciferase activity similarly to CLuc. However, because of the amino acid substitutions, the protein is characterized by an emission spectral peak of 435 nm or less and particularly an emission spectral peak ranging from 420 nm to 435 nm (e.g., 425 nm to 435 nm) when luminescence is produced upon luciferin oxidation by the luciferase activity, whereas the same of CLuc is 454 nm. Here, examples of the amino acid substitution at each amino acid position include a combination of the following (A) to (G):
(A) a substitution of methionine at position 178 with arginine;
(B) a substitution of leucine at position 191 with glutamine;
(C) a substitution of tyrosine at position 280 with aspartic acid;
(D) a substitution of arginine at position 372 with leucine;
(E) a substitution of glutamine at position 403 with proline;
(F) a substitution of asparagine at position 404 with glycine; and
(G) a substitution of threonine at position 405 with methionine.
Meanwhile, the mutant luciferase according to (b) above among the 9th mutant luciferases consists of an amino acid sequence that has a deletion, a substitution, or an addition of one or several (e.g., 1 to 10, preferably 1 to 5, and particularly preferably 1 to 3) amino acids at positions other than the predetermined positions of the above amino acids with respect to the mutant luciferase according to (a) and has luciferase activity with an emission spectral peak of 435 nm or less. Examples of the positions other than the predetermined positions of the above amino acids include methionine at position 291 and threonine at position 313.
Furthermore, the mutant luciferase according to (c) above among the 9th mutant luciferases is a mature protein from which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (a).
Furthermore, the mutant luciferase according to (d) above among the 9th mutant luciferases is a mature protein from which a secretory signal peptide of CLuc has been removed from the amino acid sequence of the mutant luciferase according to (b).
In addition, the mutant luciferases according to the present invention further include a protein that maintains each predetermined amino acid substitution, consists of an amino acid sequence having 80% or more, preferably 90% or more, and particularly preferably 95% or more amino acid identity with respect to the amino acid sequence of the protein according to (a) or (c) of the above-described 1st to the 9th mutant luciferases, and has luciferase activity with a predetermined emission spectral peak.
Furthermore, concerning the emission spectral peaks of luciferases, errors may occur due to a measurement method, a spectrum correction method, smoothing, or the like employed. Hence, the mutant luciferases according to the present invention also includes a mutant luciferase having luciferase activity with an emission spectral peak within an error range of several nm (e.g., ±5 nm, preferably ±4 nm, and particularly preferably ±2 nm) with respect to the above emission spectral peak, as long as the above relative emission spectral peak shifts are involved with respect to the wild-type luciferase (CLuc).
Hereinafter, the 1st to the 9th mutant luciferases are together referred to as “the mutant luciferase(s) according to the present invention.”
The above mutant luciferases according to the present invention can be prepared in the form of fusion proteins linked to foreign proteins or peptides. Here, the term “foreign protein or peptide” refers to an exogenous protein or peptide with respect to the mutant luciferases according to the present invention. Examples of foreign proteins or peptides include proteins or peptides (e.g., glutathione S-transferase, a maltose binding protein, thioredoxin, a cellulose binding domain, a streptavidin-binding peptide, and a histidine tag) to be used for protein purification, and signal peptides (e.g., a secretory signal peptide (amino acid sequence: SEQ ID NO: 3) of the α factor of budding yeast, a signal peptide (amino acid sequence: SEQ ID NO: 4) of invertase of budding yeast, and a signal peptide (amino acid sequence: SEQ ID NO: 5) of membrane protein Ste6p of budding yeast) for extracellular secretion or transfer to intracellular organs. For example, a gene encoding a fusion protein prepared by linking a secretory signal peptide appropriate for a host to be transformed to the mature protein of a mutant luciferase according to the present invention is transformed into the host, so that the mutant luciferase according to the present invention can be secreted and expressed extracellularly. A position in such a mutant luciferase according to the present invention, to which a foreign protein or peptide is linked, can be adequately selected so that the mutant luciferase according to the present invention and a foreign protein or peptide can each retain its functions or activity. For example, in the case of a fusion protein prepared by linking a secretory signal peptide to the mature protein of a mutant luciferase according to the present invention, the secretory signal peptide can be linked to the N-terminal side of the mature protein (specifically, the N-terminal side of amino acid at position 19 of the amino acid sequence shown in SEQ ID NO: 2).
The genes according to the present invention are genes encoding the mutant luciferases according to the present invention or genes encoding the above fusion proteins. The mutant luciferases according to the present invention or the fusion proteins can be expressed via introduction of these genes into hosts.
Hosts are not particularly limited. Examples of hosts include yeast, bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Bacillus such as Bacillus subtilis, or bacteria of the genus Pseudomonas such as Pseudomonas putida, animal cells such as COS cells, insect cells such as Sf9, or plants belonging to the family Brassicaceae. Yeast may be any yeast and examples of yeast include Saccharomyces cerevisiae, Shizosaccharomyces pombe, Pichia pastoris, Candida albicans, and Hansenula polymorpha. In particular, Saccharomyces cerevisiae is preferred.
First, the genes encoding the mutant luciferases according to the present invention or the genes encoding foreign proteins or peptides are prepared. These genes can be easily obtained by PCR using, for example, genomic DNA or the like of an organism (e.g., Cypridina noctiluca) from which the genes are derived as a template and primers complementary to the nucleotide sequences at both ends of the relevant region. However, the mutant luciferases according to the present invention have amino acid substitutions with respect to the amino acid sequence of CLuc. Thus, the genes encoding the mutant luciferases according to the present invention can be obtained by further introducing mutations into the PCR products obtained as described above by site-directed mutagenesis or the like.
Once the nucleotide sequences are confirmed, genes encoding the mutant luciferases according to the present invention or genes encoding foreign proteins or peptides can then be obtained by chemical synthesis, PCR using cloned probes as templates, or hybridization using DNA fragments having the nucleotide sequences as probes. Furthermore, mutants which are derived from genes encoding the mutant luciferases according to the present invention or genes encoding foreign proteins or peptides and have functions equivalent to those before mutation can be synthesized by site-directed mutagenesis or the like.
In addition, for introduction of mutations into genes encoding the mutant luciferases according to the present invention or genes encoding foreign proteins or peptides, a known technique such as the Kunkel method or the Gapped duplex method or a method according thereto can be employed. For example, mutations are introduced using a mutagenesis kit (e.g., Mutant-K (TAKARA) or Mutant-G (TAKARA)) using site-directed mutagenesis or a LA PCR in vitro Mutagenesis series kit (TAKARA).
When a gene encoding a fusion protein is prepared by ligating a gene encoding the mutant luciferase according to the present invention to a gene encoding a foreign protein or peptide, DNA is prepared by ligating the gene encoding the foreign protein or peptide to the gene encoding the mutant luciferase according to the present invention. Such DNA itself may be prepared via ligation, a vector containing the DNA, or the like.
As a method for ligating a gene encoding a foreign protein or peptide to a gene encoding a mutant luciferase according to the present invention, a method is employed, which involves cleaving each purified gene encoding the mutant luciferase according to the present invention and a purified gene encoding a foreign protein or peptide with an appropriate restriction enzyme and then ligating the genes to each other. Furthermore, a method that may be employed herein involves providing a homologous region in a gene encoding a mutant luciferase according to the present invention and a gene encoding a foreign protein or peptide and then ligating them by an in vitro method using PCR or the like or an in vivo method using yeast or the like.
A recombinant vector containing a gene according to the present invention can be obtained by inserting the gene according to the present invention into an appropriate vector. Vectors that can be used herein are not particularly limited, as long as they are capable of replicating within hosts. Examples of such vectors include plasmids, shuttle vectors, and helper plasmids. When a vector is incapable of replicating, the vector may be a DNA fragment that will become capable of replicating when it is inserted into the chromosome of a host, for example.
Examples of plasmid DNA include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, and pBluescript), plasmids derived from Bacillus subtilis (e.g., pUB110 and pTP5), and plasmids derived from yeast (e.g., the YEp line such as YEp13 and the YCp line such as YCp50). Examples of phage DNA include λ phages (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP). Furthermore, animal viruses such as a retrovirus or a vaccinia virus and vectors of insect viruses such as a baculovirus can also be used.
A method for inserting the gene according to the present invention into a vector can be carried out in accordance with a method that involves ligating a gene encoding a foreign protein or peptide to a gene encoding a mutant luciferase according to the present invention, as described above.
Moreover, a transformant is prepared by introducing a gene according to the present invention or a recombinant vector (hereinafter, referred to as “the recombinant vector or the like according to the present invention”) containing the gene according to the present invention into a host.
A method for introducing the recombinant vector or the like according to the present invention into yeast is not particularly limited, as long as the method is used for introducing DNA into yeast. Examples of the method include electroporation, a spheroplast method, and a lithium acetate method. Moreover, a yeast transformation method that may also be used herein is a type of substitution and/or insertion into a chromosome using a DNA sequence homologous to an arbitrary region in a vector (e.g., YIp vector) or a chromosome. Furthermore, a method for introducing the recombinant vector or the like according to the present invention into yeast may be any method described in general experimental protocols, journal articles, or the like.
A method for introducing the recombinant vector or the like according to the present invention into bacteria is not particularly limited, as long as it is a method for introducing DNA into bacteria. Examples of the method include a method using calcium ions and electroporation.
When animal cells are used as hosts, monkey cells (COS-7 and Vero), Chinese hamster ovary cells (CHO cells), mouse L cells, and the like are used. Examples of a method for introducing the recombinant vector or the like according to the present invention into animal cells include electroporation, a calcium phosphate method, and lipofection.
When insect cells are used as hosts, Sf9 cells and the like are used. Examples of a method for introducing the recombinant vector or the like according to the present invention into insect cells include a calcium phosphate method, lipofection, and electroporation.
When plants are used as hosts, whole plant bodies, plant organs (e.g., leaves, petals, stems, roots, and seeds), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, and vascular bundle), cultured plant cells, and the like are used. Examples of a method for introducing the recombinant vector or the like according to the present invention into plants include electroporation, an agrobacterium method, a particle gun method, and the PEG method.
Whether or not the recombinant vector or the like according to the present invention has been incorporated into hosts can be confirmed by the PCR method, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformant, DNA-specific primers are designed, and then PCR is carried out. Subsequently, the amplification products are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and then stained with ethidium bromide, SYBR Green solution, or the like. The amplification products are detected as bands, so that transformation is confirmed. Furthermore, PCR is carried out using primers previously labeled with a fluorescent dye or the like, and then amplification products can also be detected. Furthermore, a method that may also be employed herein involves binding amplification products to a solid phase such as a microplate and then confirming the amplification products by fluorescence reaction, enzyme reaction, or the like.
Next, the thus obtained transformant is cultured under conditions that enable the growth. When the culture or culture supernatant of the transformant is directly subjected to measurement of enzyme activity, the transformant is cultured under conditions where the mutant luciferases according to the present invention are not deactivated. For example, when transformed yeast in which the recombinant vector or the like according to the present invention has been introduced is cultured, the temperature is set ranging from 4° C. to 37° C. and preferably ranging from 20° C. to 30° C., for example, so that yeast can grow and the mutant luciferases according to the present invention are not deactivated. Furthermore, pH for medium is set ranging from 3.5 to 6.5 and preferably ranging from 5.5 to 6.0, for example. Time for culturing ranges from 1 to 120 hours and preferably ranges from 1 to 24 hours corresponding to the logarithmic growth phase, for example.
As described above, the mutant luciferases according to the present invention or fusion proteins of the mutant luciferase according to the present invention with foreign proteins or peptides can be obtained from the above transformant.
When the activity of the mutant luciferases according to the present invention is measured, for example, the above transformants are cultured and then the thus obtained culture or culture supernatant is caused to come into contact with luciferin, that is a substrate, (e.g., sea-firefly luciferin) or a derivative thereof under conditions where the enzyme reaction of the mutant luciferases according to the present invention can take place. Here, an example thereof is a luciferin derivative in which the chemical structure of a side chain at position C2, C6, or C8 in the imidazopyrazinone skeleton of the luciferin has been substituted with a functional group (e.g., an aromatic group, aliphatic group, carboxylic acid, or amino group) or the like that is electrolyzed in an aqueous solution. The structure or position of a functional group is not limited, as long as light is emitted (luminescence is produced) by the functions of sea-firefly luciferase. Because of such substitution, enhancement in luminescence intensity, improvement of suppression of autolysis, and the like can be expected.
Moreover, conditions where enzyme reaction takes place mean conditions where luciferin specifically binds to the active center of the mutant luciferase according to the present invention so as to generate a complex and thus the enzyme reaction proceeds. Moreover, the phrase “contact ( . . . to come into contact with . . . )” refers to a condition where the mutant luciferases according to the present invention in cultures or culture supernatants to come close to luciferin, so that enzyme reaction takes place. Furthermore, the term “culture(s)” refers to a culture solution or a medium containing a transformant. For example, when a mutant luciferase according to the present invention is linked to a secretory signal peptide appropriate for a host, the mutant luciferase is secreted in the medium. Therefore, such a culture solution or medium containing a transformant can be used intact. Alternatively, a culture supernatant obtained by separation of a transformant by centrifugation or the like can also be used. Such a culture supernatant can also be subjected to dilution, condensation, dialysis, purification, or the like.
Regarding conditions for causing contact, the temperature is set at 0° C. to 40° C. and preferably 15° C. to 30° C., for example. Furthermore, the pH is set at 4.0 to 9.0 and preferably 6.0 to 8.0, for example. The contact time (reaction time) ranges from 1 second to 30 minutes and preferably ranges from 1 second to 30 seconds, for example. In particular, a solution of luciferin or a derivative thereof, which has been diluted with any one of various buffers is added to a culture or a culture supernatant, so that the pH of the culture or the culture supernatant can be shifted to the pH at which the enzyme activity of the mutant luciferase(s) according to the present invention is high. For example, the above-described pH upon contact can be set by adding to a culture or a culture supernatant containing the mutant luciferase(s) according to the present invention the solution of luciferin or a derivative thereof, which is prepared via dilution with a buffer, such as Tris hydrochloric acid buffer (Tris-HCl) that is 2 M or less (preferably ranges from 50 mM to 200 mM) and has pH ranging from 3.5 to 9.0 (preferably, pH 7.0 to pH 8.0).
Regarding the concentration of luciferin or a derivative thereof, which is a substrate for a culture or a culture supernatant, for example, luciferin or a derivative thereof is added to a final concentration of 0.1 μM or more and preferably ranging from 1.25 μM to 2.5 μM, with respect to 0.05 or more of the turbidity (e.g., absorbance at 600 nm) of the culture or the culture supernatant of a transformant having the mutant luciferase(s) according to the present invention.
Next, the enzyme activity of the mutant luciferases according to the present invention is measured. For example, a measurement method involves subjecting a mixture of the culture or the culture supernatant of a transformant with luciferin or a derivative thereof to luminescence measurement using a luminometer and then measuring the enzyme activity as relative light unit (RLU). Moreover, to standardize a measured value by correcting the enzyme activity upon activity measurement, turbidity (e.g., absorbance at 600 nm) of a culture solution or a culture supernatant is measured and then the relative light unit is divided by turbidity for correction, so that the thus corrected value (RLU/OD) can be obtained as an enzyme activity level. Alternatively, a method that is also preferable for standardization of relative light unit involves measuring the level of ATP contained in a transformant and then dividing the relative light unit by the ATP level. Furthermore, another method that may also be employed herein involves causing simultaneous expression of another enzyme or protein in the transformant, measuring the enzyme or protein amount, and then dividing the relative light unit by the level for correction. The mutant luciferases according to the present invention exert luciferase activity with the emission spectral peaks differing from that of CLuc. With another method that may also be employed herein, luminescence can be distinguished from the other using such characteristics of having different emission spectra. The method involves causing expression of CLuc and then dividing by the luminescence derived from CLuc for correction.
Furthermore, for example, when a host is a microorganism such as Saccharomyces cerevisiae, a transformant is grown in agar medium to form colonies. Hence, for example, enzyme activity can be measured by adding luciferin or a derivative thereof to agar medium containing a transformant and then measuring the luminescence intensity of the colonies using a luminescence detector having a CCD camera, for example.
Furthermore, the mutant luciferases according to the present invention exert luciferase activity with emission spectral peaks differing from that of CLuc. Hence, in addition to the above measurement of luciferase activity, whether or not the mutant luciferases according to the present invention have emission spectral peaks within the above range is confirmed by measurement using a luminescence detector with a plurality of optical filters that have different transmission characteristics and a CCD camera, for example.
When significant luciferase activity and expected emission spectral peaks are confirmed through measurement of such luciferase activity and emission spectral peaks, it can be concluded that the mutant luciferases according to the present invention have been obtained.
Furthermore, according to the above-explained measurement of emission spectral peaks, the transcriptional activity of a plurality of promoters can be evaluated simultaneously using CLuc and the mutant luciferases according to the present invention as reporter proteins.
In the method for evaluating the transcriptional activity of promoters according to the present invention, 2 or more luciferases are used from among wild-type CLuc, the mutant luciferases according to the present invention, and fusion proteins of wild-type CLuc or the mutant luciferases according to the present invention with foreign proteins or peptides. Here, the term “wild-type CLuc” refers to the following protein (a) or (b):
(a) CLuc consisting of the amino acid sequence shown in SEQ ID NO: 2; or
(b) a mature protein consisting of an amino acid sequence that has a deletion of a secretory signal peptide with respect to the amino acid sequence shown in SEQ ID NO: 2.
Moreover, the term “a fusion protein(s) of wild-type CLuc with a foreign protein(s) or peptide(s)” refers to a fusion protein in which a foreign protein or peptide is linked to the protein (a) or (b) above.
First, DNA in which different promoters to be evaluated have been ligated to the 5′ upstream side of each of these two or more luciferase genes is introduced into a host. Ligation of a promoter to the 5′ upstream side of the luciferase gene results in arrangement of the luciferase gene under the control of the promoter. Subsequently, the thus obtained transformant is cultured and then a culture or a culture supernatant is obtained. Furthermore, the culture or the culture supernatant is caused to come into contact with luciferin or a derivative thereof. Next, different luminescence intensities due to different emission spectral peaks resulting from the activity of a plurality of luciferases introduced are measured, so that the transcriptional activity of a plurality of promoters can be evaluated simultaneously and quantitatively. At this time, based on the transcriptional activity of one of the plurality of promoters, the transcriptional activity of the other promoters can be corrected. Different luminescence intensities due to a plurality of different emission spectral peaks can be measured by installing an appropriate filter set in a luminometer applicable for multi-reporter assays, “AB-2250 Luminescensor MCA (ATTO Corporation),” for example, which is an apparatus for which the principle according to JP Patent Publication (Kokai) No. 2004-187652 has been applied.
Moreover, the mutant luciferases according to the present invention or fusion proteins of the mutant luciferases according to the present invention with foreign proteins or peptides are used for BRET (Bioluminescence resonance energy transfer) or the like, so that light emission or energy release can be caused.
In the method for emitting light or releasing energy according to the present invention, first, the mutant luciferases according to the present invention or fusion proteins of the mutant luciferases according to the present invention with foreign proteins or peptides is come into contact with luciferin or a derivative thereof. Because of this contact, luciferin is oxidized to oxyluciferin in its excited state. Subsequently, the oxyluciferin in its excited state and a chemical substance are caused to act on each other. Here, the term “chemical substance” refers to a substance that receives excitation energy of a luminous object via energy resonance so as to be able to produce fluorescence because of the energy. Examples of such chemical substance include fluorescein, FITC, TRITC, TAMRA, and fluorescent proteins such as GFP (green fluorescent protein derived from Aequorea victoria) and mutants thereof (e.g., CFP and YFP), and DsRed (red fluorescent protein derived from Porifera). Furthermore, the expression “to act.” refers to arrangement of oxyluciferin and a chemical substance at positions that enable (in terms of distance) topological energy transfer.
The oxyluciferin and the chemical substance are then caused to act on each other, so that luminescence or energy involved in luminescence produced when the oxyluciferin returns to the ground state shifts to the chemical substance. The chemical substance is excited so as to be able to emit light or release energy depending on the excitation energy.
As explained above, the use of the mutant luciferases according to the present invention makes it possible to measure (multi-reporter assay) the transcriptional activity of a plurality of different promoters using a single type of substrate and a single time of luminescence measurement. Moreover, with the use of the mutant luciferases according to the present invention, an emission spectrum corresponding to the excitation spectrum of a specific chemical substance is provided, so that higher BRET efficiency is exerted and a strong signal can be obtained. Furthermore, the use of the mutant luciferases according to the present invention makes it possible to conduct simultaneous analysis of structural changes of a plurality of proteins using a plurality of BRETs.
EXAMPLES
Hereinafter, the present invention will be described in detail by examples as follows, but the scope of the present invention is not limited by these examples.
The plurality of mutant luciferases according to the present invention and the luciferase from which the mutant luciferases are derived were expressed in Saccharomyces cerevisiae and the resulting emission spectra were compared.
Example 1
Secretion and Expression of CLuc in Saccharomyces cerevisiae 
The plasmid pCLuRA-TDH3 disclosed in the pamphlet of International Publication No. 2006/132350 was used as an expression vector for secretion and expression of CLuc in Saccharomyces cerevisiae. 
The plasmid pCLuRA-TDH3 contained a gene (hereinafter, referred to as “αCLuc gene”) encoding a fusion protein (hereinafter, referred to as “αCLuc”) of a secretory signal peptide (amino acid sequence: SEQ ID NO: 3) of α factor of budding yeast with a CLuc mature protein (the amino acid sequence resulting when an amino acid sequence ranging from positions 1 to 18 had been removed from the amino acid sequence of CLuc shown in SEQ ID NO: 2). The amino acid sequence shown in SEQ ID NO: 6 was the amino acid sequence of αCLuc. The fusion protein of CLuc with the secretory signal peptide derived from α factor was prepared, so that CLuc was secreted outside the microorganisms.
Furthermore, in the plasmid pCLuRA-TDH3, a promoter of the Saccharomyces cerevisiae TDH3 (systematic gene name: YGR192C) gene had been incorporated upstream (5′ side) of the αCLuc gene. The expression of the αCLuc gene is controlled by the promoter. The nucleotide sequence shown in SEQ ID NO: 7 is a partial nucleotide sequence of the plasmid pCLuRA-TDH3, containing a 700-bp 5′ upstream initiation codon of αCLuc containing a TDH3 promoter sequence, the αCLuc coding region, and a 300-bp 3′ downstream termination codon of αCLuc containing a CYC1 terminator sequence.
The Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed using the plasmid pCLuRA-TDH3. An EZ-transformation kit (BIO101) was used for transformation.
A uracil-free synthetic agar medium (0.67% yeast nitrogen base without amino acids (Becton, Dickinson and Company), 40 μg/ml adenine, 20 μg/ml L-arginine monohydrochloride, 100 μg/ml L-aspartic acid, 100 μg/ml L-sodium glutamate monohydrate, 20 μg/ml L-histidine, 60 μg/ml L-leucine, 30 μg/ml L-lysine hydrochloride, 20 μg/ml L-methionine, 50 μg/ml L-phenylalanine, 375 μg/ml L-serine, 200 μg/ml L-threonine, 40 μg/ml L-tryptophan, 30 μg/ml L-tyrosine, 150 μg/ml L-valine, 2% glucose, and 2.0% agar; hereinafter, referred to simply as “SD-ura agar medium”) was coated with the thus obtained transformant, followed by 3 days of culture at 30° C. As a result, a transformant containing the plasmid pCLuRA-TDH3 was obtained.
The transformant containing the plasmid pCLuRA-TDH3 obtained as described above was inoculated into a uracil-free synthetic liquid medium (0.67% yeast nitrogen base without amino acids (Becton, Dickinson and Company), 40 μg/ml adenine, 20 μg/ml L-arginine monohydrochloride, 100 μg/ml L-aspartic acid, 100 μg/ml L-sodium glutamate monohydrate, 20 μg/ml L-histidine, 60 μg/ml L-leucine, 30 μg/ml L-lysine hydrochloride, 20 μg/ml L-methionine, 50 μg/ml L-phenylalanine, 375 μg/ml L-serine, 200 μg/ml L-threonine, 40 μg/ml L-tryptophan, 30 μg/ml L-tyrosine, 150 μg/ml L-valine, 2% glucose, and 200 mM potassium phosphate, pH 6.0; hereinafter, referred to simply as “buffered SD-ura medium”) having buffering action, followed by 24 hours of shake culture at 30° C.
After shake culture, a culture solution was centrifuged, so that a culture supernatant was isolated. Eighty (80) μl of a luciferin solution (1 μM luciferin, 100 mM Tris-HCl, pH 7.4) was added to 20 μl of the thus isolated culture supernatant and then luminescence was measured using a Berthold LB960 luminometer. As a result, luminescence was observed at 4×105 RLU/second. Specifically, secretion and expression of CLuc in the Saccharomyces cerevisiae BY4743ΔPRB1 strain was confirmed.
Example 2
Isolation of Mutant Luciferase Via Introduction of Random Mutation
2-1. Construction of Mutant CLuc Gene Library (N-Region Mutant Library)
A random point mutation was introduced into the αCLuc coding region of the plasmid pCLuRA-TDH3 by error prone PCR.
The subject region for introduction of the mutation was the first half portion of the αCLuc coding region (in the nucleotide sequence shown in SEQ ID NO: 7, the nucleotide sequence ranging from positions 900 to 1813; hereinafter, referred to as “N region”). The reason for limiting the range is that amplification of a long region is frequently difficult in error prone PCR. Moreover, the reason for not using as a subject region the nucleotide sequence ranging from positions 701 to 899 of the nucleotide sequence shown in SEQ ID NO: 7 is that this portion is a region encoding most of the secretory signal peptide of a factor.
The following oligo DNA primers were used in error prone PCR of the N region.









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 of the N region is as follows: Taq DNA polymerase (Roche, 1 unit/μl): 5 μl; 10×PCR buffer without magnesium ion: 10 μl; mixed solution of deoxynucleotide for error prone PCR: 10 μl; 25 mM magnesium chloride: 28 μl; 5 mM manganese chloride: 2.5 μl; plasmid pCLuRA-TDH3 solution (150 ng/μl): 1 μl; mut-Cluc-F (SEQ ID NO: 8) (10 pmol/μl): 3 μl; mut-CLuc-NR2 (SEQ ID NO: 9) (10 pmol/μl): 3 μl; and sterile water: 37.3 μl.
The composition of the above mixed solution of deoxynucleotide for error prone PCR is as follows: 100 mM dCTP: 100 μl; 100 mM dTTP: 100 μl; 100 mM dGTP: 20 μl; 100 mM dATP: 20 μl; and sterile water: 760 μl.
Error prone PCR was carried out for 30 cycles each consisting of 94° C. for 1 minute (denaturation), 45° C. for 1 minute (annealing), and 72° C. for 1 minute (extension).
The PCR product obtained by error prone PCR was electrophoresed with 1% agarose, so that an approximately 900-bp DNA fragment was confirmed. This fragment was purified using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), thereby preparing a DNA solution.
Next, to ensure the obtainment of DNA in a sufficient amount for introduction into Saccharomyces cerevisiae, PCR was further carried out using the above DNA solution as a template (hereinafter, referred to as “2nd PCR”).
The composition of the reaction solution of the 2nd PCR is as follows: KOD plus DNA polymerase (TOYOBO Co., Ltd.): 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; mut-Cluc-F (SEQ ID NO: 8) (10 pmol/μl): 1.5 μl; mut-CLuc-NR2 (SEQ ID NO: 9) (10 pmol/μl): 1.5 μl; the above DNA solution: 1 μl; and sterile water: 33 μl.
The 2nd PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 50° C. for 30 seconds (annealing), and 68° C. for 1 minute (extension).
The PCR product obtained by the 2nd PCR was electrophoresed with 1% agarose, so that an approximately 900-bp DNA fragment was confirmed. The DNA fragment is hereinafter referred to as “N-region fragment.”
The N-region fragment was purified from agarose gel using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer (hereinafter, referred to as “N-region fragment solution”).
Saccharomyces cerevisiae generally undergoes homologous recombination within cells with high probability. Accordingly, a linear DNA fragment (hereinafter, referred to as a “complementary N-region fragment”) lacking the nucleotide sequence ranging from positions 967 to 1703 of the nucleotide sequence as shown in SEQ ID NO: 7 in the nucleotide sequence of the plasmid pCLuRA-TDH3 and the “N-region fragment” in which a mutation has been introduced as described above are simultaneously introduced into Saccharomyces cerevisiae. Within Saccharomyces cerevisiae, circular DNA (the mutant plasmid pCLuRA-THD3 wherein a mutation has been introduced into the N region) is reconstituted by homologous recombination, so that Saccharomyces cerevisiae can be transformed with the reconstituted plasmid.
The “complementary N-region fragment” was prepared by PCR as follows. The following oligo DNA primers were used for PCR.









vec-CLuc-R:




GCTTCAGCCTCTCTTTTCTCGAGAG
(SEQ ID NO: 10)


 


SQ-CLuc-NF2:


TTCTCGAGCCGTACAAGGACAGCTGCCGCA
(SEQ ID NO: 11)






The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase (TOYOBO Co., Ltd.): 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; vec-CLuc-R (SEQ ID NO: 10) (10 pmol/μl): 1.5 μl; SQ-CLuc-NF2 (SEQ ID NO: 11) (10 pmol/μl): 1.5 μl; plasmid pCLuRA-TDH3 solution (150 ng/μl): 1 μl; and sterile water: 33 μl.
PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation) and 68° C. for 8 minutes (annealing and extension).
The thus obtained PCR product was electrophoresed with 1% agarose, so that an approximately 7-kbp DNA fragment was confirmed. The DNA fragment was purified using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer (hereinafter, referred to as a “complementary N-region fragment solution”).
The overlapped portions between the N-region fragment and the complementary N-region fragment were the nucleotide sequence ranging from positions 900 to 966 and the nucleotide sequence ranging from positions 1704 to 1813 of the nucleotide sequence shown in SEQ ID NO: 7.
Five (5) μl of the N-region fragment solution was mixed with 5 μl of the complementary N-region fragment solution. The Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed by the lithium acetate method. An SD-ura agar medium was coated with the Saccharomyces cerevisiae BY4743ΔPRB1 strain subjected to transformation, followed by incubation at 30° C. for 48 hours. Many colonies that thus appeared were used as N-region mutant libraries.
2-2. Construction of Mutant CLuc Gene Library (C-Region Mutant Library)
In a manner similar to that used for the above construction of the N-region mutant libraries, C-region mutant libraries were constructed using the last half portion of the αCLuc coding region as the subject region for introduction of mutation.
The subject regions for introduction of mutation were the last half portion of the αCLuc coding region and an approximately 60-bp 3′ non-coding region in the αCLuc coding region (the nucleotide sequence ranging from positions 1554 to 2663 of the nucleotide sequence shown in SEQ ID NO: 7; hereinafter, referred to as the “C region”). The reason why the 3′ non-coding region of the αCLuc coding region was contained in the C region is to cause intracellular homologous recombination to take place outside the αCLuc coding region into which mutation should have been introduced and thus not to affect the mutagenesis efficiency with respect to the C-terminal coding region in the αCLuc coding region.
A C-region fragment corresponding to the N-region fragment of the above N-region mutant libraries was prepared by carrying out PCR in a manner similar to that used for error prone PCR and the 2nd PCR above except that the following oligo DNA primers were used.









mut-CLuc-CF1:




TCTCTGGCCTCTGTGGAGATCTTAAAATGA
(SEQ ID NO: 12)


 


mut-CLuc-R:


AACTCCTTCCTTTTCGGTTAGAGCGGATGT
(SEQ ID NO: 13)






The thus obtained PCR product was electrophoresed with 1% agarose, so that an approximately 1,100-bp DNA fragment was confirmed. Next, the DNA fragment (C-region fragment) was purified from agarose gel using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer, thereby resulting in a C-region fragment solution.
A complementary C-region fragment corresponding to the complementary N-region fragment of the above N-region mutant libraries was prepared. The complementary C-region fragment was prepared according to the method for preparing a complementary N-region fragment, except that the following oligo DNA primers and PCR reaction conditions were used.
Oligo DNA primers used herein are as follows.









vec-CLuc-F:




TCTAGAGGGCCGCATCATGTAATTA
(SEQ ID NO: 14)


 


SQ-CLuc-CR1:


TGGACAACCGTCAAACTCCTGGTTGATCTT
(SEQ ID NO: 15)






PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 55° C. for 30 seconds (annealing), and 68° C. for 8 minutes (extension).
The thus obtained PCR product was electrophoresed with 1% agarose, so that an approximately 6.5-kbp DNA fragment was confirmed. This DNA fragment (complementary C-region fragment) was purified using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), subjected to ethanol precipitation, and then dissolved in 20 μl of TE buffer, thereby resulting in a complementary C-region fragment solution.
The overlapped portions between the C-region fragment and the complementary C-region fragment were the nucleotide sequence ranging from positions 1554 to 1663 and the nucleotide sequence ranging from positions 2576 to 2663 of the nucleotide sequence shown in SEQ ID NO: 7.
Five (5) μl of the C-region fragment solution was mixed with 5 μl of the complementary C-region fragment solution. The Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed by the lithium acetate method. An SD-ura agar medium was coated with the Saccharomyces cerevisiae BY4743ΔPRB1 strain subjected to transformation, followed by incubation at 30° C. for 48 hours. Many colonies that thus appeared were used as C-region mutant libraries.
2-3. Screening for Mutant Luciferases
Mutant luciferases with emission spectra that had shifted were screened for by filming sequentially the fluorescence using a CCD camera and 2 types of optical filter with different transmission characteristics. The optical filters used herein are GG495 and BG28 (SCHOTT). The former filter is a long-pass filter with a cut-off wavelength of around 495 nm. The latter filter is a band-pass filter with a maximum transmission wavelength of around 450 nm. The same sample (culture supernatant containing luciferase) was filmed with a CCD camera using these filters successively and then the recorded signal intensities were compared. Differences between the resulting ratio and that of wild-type CLuc indicate the occurrence of an emission spectral shift.
One (1) ml each of a buffered SD-ura medium was dispensed into a 96-well deep well plate (2 ml/well), and colonies of the N-region mutant libraries or the C-region mutant libraries were inoculated in separate wells (one colony per well) using a colony picker. In addition, as a control, Saccharomyces cerevisiae (BY4743ΔPRB1 strain transformed with plasmid pCLuRA-TDH3) secreting and expressing wild-type Cluc (into which no mutation had been introduced) was inoculated into 6 wells.
Subsequently, the inoculated plate was subjected to approximately 48 hours of culture at 30° C., centrifugation was carried out at 1,800 rpm, and then 20 μl of the culture supernatant was transferred from each well to a black 96-well plate.
A luciferin solution (1 μM luciferin, 100 mM Tris-HCl, pH 7.4) was added to each well, the plate was then set within a Light Capture (ATTO) provided with GG495, and then filming was carried out for 30 seconds to 2 minutes. Next, the optical glass filter was exchanged with BG28 immediately after filming, and filming was then carried out again for 30 seconds to 2 minutes. The thus filmed images were stored in the form of TIFF files in a computer.
Images filmed with the use of each filter were processed by image processing software (e.g., Adobe Photoshop). The ratio of the signal intensity of the image filmed with GG495 to the same of the image filmed with BG28 was subjected to pseudo-color processing and then the result was visually compared with that for wild-type Cluc. Therefore, clones suspected of having undergone emission spectral shift were selected.
As described above, as a result of screening of one thousand and hundreds of clones in each of the N-region mutant library and the C-region mutant library, an M178K mutant (a clone having M178K mutant CLuc in which methionine at position 178 of the amino acid sequence shown in SEQ ID NO: 2 had been substituted with lysine; corresponding to a transformant having the 2 mutant luciferase(s)) was obtained from the N-region mutant library, in which a shift to the shorter wavelength side was thought to take place; and a K375R mutant and a K375E mutant (a clone having K375R mutant having a substitution of lysine at position 375 of the amino acid sequence shown in SEQ ID NO: 2 with arginine and a clone having K375E mutant Cluc having a substitution of the same with glutamic acid; corresponding to a transformant having the 11 mutant luciferase(s)) were obtained from C-region mutant library, in which a shift to the longer wavelength side was thought to take place.
Here, for example, the term “K375R mutant Cluc” represents the mutant Cluc that has a substitution of lysine corresponding to position 375 of SEQ ID NO: 2 with arginine. The alphabetical letter representing an amino acid is a single letter selected based on the recommendation of the International Union of Pure and Applied Chemistry—International Union of Biochemistry (IUPAC-IUB). Moreover, the term “K375R mutant” refers to a clone having K375R mutant CLuc. Furthermore, a plasmid retained by the K375R mutant is referred to as “pCLuRA-TDH3[K375R].” Hereinafter, mutant CLuc, a mutant (clone) having mutant CLuc, and a plasmid retained by a mutant are designated in a similar manner.
2-4. Measurement of Emission Spectrum of Mutant Luciferase
The following transformed yeast (a) to (d) was separately shake-cultured in a buffered SD-ura medium and then centrifuged, so as to allow collection of the culture supernatant. The thus collected culture supernatants were each concentrated about 10 fold with a VivaSpin (molecular weight cut off: 10,000, SARTORIUS K. K.).
(a) Saccharomyces cerevisiae (the BY4743ΔPRB1 strain transformed with plasmid pCLuRA-TDH3) secreting and expressing wild-type CLuc
(b) M178K mutant
(c) K375R mutant
(d) K375E mutant
Next, the thus obtained concentrated solution of each culture supernatant was subjected to AB-1850 LumiFLSpectroCapture (emission spectrophotometer) (ATTO) so as to measure the emission spectra.
The composition of the reaction solution is as follows: 1 μM luciferin, 100 mM Tris-HCl, pH 7.5, and the above concentrated solution (approximately 1 μl to 3 μl).
The measured emission spectra are shown in FIG. 1. FIG. 1 shows the relative light unit for each luciferase to the wavelength, wherein “wild-type” denotes the measurement results for wild-type CLuc, “M178K” denotes the same for M178K mutant CLuc, “K375R” denotes the same for K375R mutant CLuc, and “K375E” denotes the same for K375E mutant CLuc.
As understood from FIG. 1, in the case of visual observation, whereas the emission spectral peak of wild-type CLuc was 453 nm, the emission spectral peak of K375R mutant CLuc was 461 nm, indicating a shift to the longer wavelength side of 8 nm. Furthermore, the emission spectral peak of K375E mutant CLuc was 460 nm, indicating a shift to the longer wavelength side of 7 nm. On the other hand, the emission spectral peak of M178K mutant CLuc was 447 nm, indicating a shift to the shorter wavelength side of 6 nm.
As described above, the difference between K375R mutant CLuc and M178K mutant CLuc in terms of emission spectral peak was 14 nm and the difference between K375E mutant CLuc and M178K mutant CLuc in terms of emission spectral peak was 13 nm. Hence, it was considered that they can be used for dual reporter assays.
2-5. Determination of Spectral Peak Wavelength from the Emission Spectra of Mutant Luciferase
Regarding spectra measured using AB-1850 LumiFLSpectroCapture (emission spectrophotometer) (ATTO), data were further processed so as to find spectral peaks as follows.
Data comprising the measured wavelengths and luminescence intensities were recorded in a file using the control program attached to the instrument. Next, the recorded data was read by a macro file for correction attached to the instrument (Excel file (Microsoft)), so that the wavelength-dependent sensitivity of the detector was corrected and data were obtained from which the background (obtained by measuring the emission spectrum for the container alone) had been subtracted. Next, the data were recorded in the csv format (comma-deliminated text format) file in which the first line denotes wavelength and the second line denotes (normalized) luminescence intensity.
Furthermore, the thus obtained file was read by digital data analysis software OriginPro v7.5 (OriginLab) and then processed to remove the noise of luminescence intensities. For noise processing, FFT (fast Fourier transformation) analysis was employed. First, FFT analysis was conducted and then the wavelength distribution (in addition, since the horizontal axis on OriginPro is regarded as representing frequency, internal processing is carried out using Hz) was found. Based on the wavelength distribution, harmonic wavelength contents were regarded as noise, LPF (low-pass filter) processing was carried out, and then data filtering was carried out. Regarding the filtering of periodic wavelengths, in view of agreement between the original data containing noise and the processed data, 0.05 was employed for all data. For all data, periodic wavelength components with filtering periodic wavelengths of 0.05 or more were equally cut by LPT processing on OriginPro, inverse Fourier transform was carried out with the functions of the same program, and then the data were output on a file. This processing enabled conversion into a lower-noise smooth spectral curve without changing spectral outline and peak positions.
Finally, the file was read by Microsoft Excel 2003 (Microsoft), a wavelength at which the luminescence intensity reached the maximum was automatically detected, and then the wavelength was determined to be a peak wavelength.
Spectral measurement using wild-type CLuc and each mutant CLuc described in the section 2-4 above was basically carried out twice, and the above-mentioned determination of spectral peak wavelengths was carried out separately. When measurement was carried out more than once, the mean value of the thus obtained spectral peak wavelengths was found and this value was employed. When measurement was carried out twice or more for the same sample, a shift between a spectral peak wavelength and the mean value thereof fell approximately within 1 nm.
According to such method for determination of spectral peak wavelengths, the emission spectral peak of M178K mutant CLuc in the section 2-4 above shifted from visually measured 447 nm based on FIG. 1 to 449 nm. In addition, the emission spectral peak of wild-type CLuc and the emission spectral peaks of the other mutant CLuc shifted from visually measured 453 nm to 454 nm (wild-type CLuc), shifted from visually measured 461 nm to 463 nm (K375R mutant CLuc), and shifted from visually measured 460 nm to 462 nm (K375E mutant CLuc). It was concluded that determination of spectral peak wavelengths by visual measurement results in significant errors. Hence, a method was employed as follows, involving consistently carrying out the above-mentioned data processing and automatically determining spectral peak wavelengths via data processing.
In the following Examples, the emission spectral peaks of mutant CLuc were determined based on the method for determination of spectral peak wavelengths.
In addition, in the following Examples, unless otherwise specified, explanations of amino acid positions concern the amino acid sequence shown in SEQ ID NO: 2.
Example 3
Construction of T167 Saturation Mutant Library and Screening for Mutant CLuc
As a result of screening described in Example 2, a T167I mutant (a clone having T167I mutant CLuc in which threonine at position 167 of the amino acid sequence shown in SEQ ID NO: 2 had been substituted with isoleucine; corresponding to a transformant having the 3rd mutant luciferase(s)) was obtained. A plasmid retained by the clone is referred to as “pCLuRA-TDH3[T167I].” The emission spectral peak of the mutant CLuc secreted from the T167I mutant was 458 nm as measured by the method described in the section 2-5 in Example 2, indicating a shift to the longer wavelength side of 4 nm compared with that of wild-type CLuc.
Accordingly, a mutant library (hereinafter, referred to as the “T167 saturation mutant library”) was constructed as follows, in which amino acid at position 167 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with any one of the other amino acids. Then obtainment of a mutant with a shift to the further longer wavelength side was attempted.
The T167 saturation mutant library was constructed as follows.
First, the following PCR was carried out. Oligo DNA primers used herein are FAR-F: AACCCTCACTAAAGGGAACAAAAGCTGGCT (SEQ ID NO: 16) and T238-Rev: GTACGGGTTGGCGATGATAGG (SEQ ID NO: 17). The DNA fragment obtained by the PCR corresponded to the nucleotide sequence ranging from positions 1 to 1411 in the nucleotide sequence shown in SEQ ID NO: 7. The composition of the reaction solution for the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; FAR-F (SEQ ID NO: 16) (10 pmol/μl): 0.6 μl; T238-Rev (SEQ ID NO: 17) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3 solution (3.8 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 50° C. for 30 seconds (annealing), and 68° C. for 2 minutes and 30 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 1.4-kbp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution A.”
Next, the following PCR was carried out. Oligo DNA primers used herein are T238X-Fw: CCTATCATCGCCAACCCGTACNNNATCGGCGAGGTCACCATCGCT (SEQ ID NO: 18) and 3′-UTR: GTAATACGACTCACTATAGGGCGAA (SEQ ID NO: 19). The letter “N” in the sequence refers to any one of A, T, G, and C. A saturation mutation is introduced by “NNN” in T238X-Fw (SEQ ID NO: 18) into amino acid at position 167 of SEQ ID NO: 2. The DNA fragment obtained by PCR was the nucleotide sequence ranging from positions 1391 to 2875 of the nucleotide sequence shown in SEQ ID NO: 7, in which a random mutation had been introduced into 3 nucleotides (codon corresponding to amino acid at position 167 of SEQ ID NO: 2) following nucleotide at position 1412 by the sequence “NNN” derived from T238X-Fw (SEQ ID NO: 18). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; T238X-Fw (SEQ ID NO: 18) (10 pmol/μl): 0.6 μl; 3′-UTR (SEQ ID NO: 19) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3 solution (3.8 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 59° C. for 30 seconds (annealing), and 68° C. for 2 minutes and 30 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 1.5-kbp DNA fragment was confirmed. This fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution B.”
Furthermore, the following PCR was carried out using a mixture of equivalent amounts of DNA solution A and DNA solution B as a template. The DNA fragment that could be amplified by PCR was the nucleotide sequence ranging from positions 900 to 1813 of the nucleotide sequence shown in SEQ ID NO: 7, in which a mutation had been introduced at random into 3 nucleotides (codon corresponding to amino acid at position 167 of SEQ ID NO: 2) following nucleotide at position 1412 by the sequence “NNN” existing in the DNA molecule in DNA solution B. The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; mut-CLuc-F (SEQ ID NO: 8) (10 pmol/μl): 1.5 μl; mut-CLuc-NR2 (SEQ ID NO: 9) (10 pmol/μl): 1.5 μl; DNA solution A: 0.5 μl; DNA solution B: 0.5 μl; and sterile water: 33 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 53° C. for 30 seconds (annealing), and 68° C. for 1 minute (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 900-bp DNA fragment was confirmed. The remaining PCR solution was purified by the use of GeneElute PCR Clean-Up Kit (Sigma) and ethanol precipitation and then dissolved in 25 μl of TE buffer, thereby preparing “DNA solution C.”
Next, the Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed by the method described in Example 2 using a mixture of equivalent amounts of DNA solution C and “complementary N-region fragment” DNA solution, thereby constructing a T167 saturation mutant library.
As a result of screening of the T167 saturation mutant library by the method described in Example 2, T167K mutant (a clone having T167K mutant CLuc in which threonine at position 167 of the amino acid sequence shown in SEQ ID NO: 2 had been substituted with lysine; corresponding to a transformant having the 3rd mutant luciferase(s)). The plasmid retained by the mutant is hereinafter referred to as “pCLuRA-TDH3[T167K].”
As a result of measuring emission spectra by the method described in Example 2, the emission spectral peak of T167K mutant CLuc was 459 nm, indicating a shift to the longer wavelength side of 5 nm compared with that of wild-type CLuc.
Example 4
Construction of Plasmid pCLuRA-TDH3[αP21L,K375R]
Based on the plasmid pCLuRA-TDH3[K375R], a mutation was introduced into a portion (of the αCLuc gene) encoding the secretory signal peptide (amino acid sequence: SEQ ID NO: 3) of the α factor. Thus, a new plasmid “pCLuRA-TDH3[αP21L,K375R]” was constructed. In this plasmid, in addition to K375R mutation, proline at position 21 (of SEQ ID NO: 3 and SEQ ID NO: 6) had been substituted with leucine (this is herein after referred to as the “αP21L mutation”). The secretory signal peptide of the α factor having the αP21L mutation improves the secretion amount of the protein to be secreted, which has been linked to the C-terminal side, 7 fold or more. Introduction of the αP21L mutation increased the secretion amount of luciferase, resulting in enhanced luminescence intensity. Naturally CLuc encoded by the plasmid is K375R mutant CLuc. In addition, “plasmid pCLuRA-TDH3 [αP21L]” was a plasmid in which, with respect to the αCLuc gene of pCLuRA-TDH3, nucleotide cytosine at position 762 of SEQ ID NO: 7 had been substituted with nucleotide thymine, so as to cause substitution of proline at position 21 (of SEQ ID NO: 3 and SEQ ID NO: 6) with leucine.
A method for constructing pCLuRA-TDH3[αP21L,K375R] is as follows.
First the following PCR was carried out. The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; mut-CLuc-CF1 (SEQ ID NO: 12) (10 pmol/μl): 0.6 μl; mut-CLuc-R (SEQ ID NO: 13) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3[K375R] solution (1 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 53° C. for 30 seconds (annealing), and 68° C. for 1 minute and 15 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 1-kbp DNA fragment was confirmed. This fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution D.” The fragment corresponds to a nucleotide sequence (containing a nucleotide substitution involved in K375R amino acid substitution) ranging from positions 1554 to 2663 of the nucleotide sequence shown in SEQ ID NO: 7.
Subsequently, the following PCR was carried out. The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; vec-CLuc-F (SEQ ID NO: 14) (10 pmol/μl): 0.6 μl; SQ-CLuc-CR1 (SEQ ID NO: 15) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3[αP21L] solution (1 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 seconds (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 53° C. for 30 seconds (annealing), and 68° C. for 8 minutes (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 6.5-kbp DNA fragment was confirmed. This fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution E.” The fragment corresponds to, in the nucleotide sequence of the plasmid pCLuRA-TDH3[αP21L], a nucleotide sequence lacking a region ranging from positions 1664 to 2575 of the nucleotide sequence shown in SEQ ID NO: 7.
Next, the Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed by the method described in Example 2 using a mixture of equivalent amounts of DNA solution D and DNA solution E, thereby causing colony formation. A DNA fragment contained in DNA solution D and the same contained in DNA solution E share the nucleotide sequence ranging from positions 1554 to 1663 and the nucleotide sequence ranging from positions 2576 to 2663 of the nucleotide sequence shown in SEQ ID NO: 7.
One of the thus obtained colonies was cultured and then DNA containing the plasmid was extracted and purified from the microorganisms. Escherichia coli DH5α was transformed using the DNA and then caused to undergo colony formation. One of the thus obtained colonies was cultured and then plasmid DNA was extracted and purified by a conventional method. The nucleotide sequence ranging from positions 1 to 2875 of the nucleotide sequence shown in SEQ ID NO: 7 was examined. Thus, the occurrence of a desired nucleotide substitution was confirmed, thereby preparing pCLuRA-TDH3[αP21L,K375R].
Example 5
T405I Mutant CLuc
A new mutant CLuc gene library was constructed by the method described in Example 2. However, as a template for PCR, pCLuRA-TDH3[αP21L] was used instead of plasmid pCLuRA-TDH3. As a result of screening by the method described in Example 2, a clone (corresponding to a transformant having the 5th mutant luciferase) was obtained, having T405I mutant CLuc in which threonine at position 405 had been substituted with isoleucine with respect to the amino acid sequence shown in SEQ ID NO: 2.
As a result of measuring emission spectra by the method described in Example 2, the emission spectral peak of T405I mutant CLuc was 458 nm, indicating a shift to the longer wavelength side of 4 nm compared with that of wild-type CLuc. The plasmid retained by the T405I mutant is hereinafter referred to as “pCLuRA-TDH3[αP21L,T405I].”
Example 6
CLuc Labeled with Histidine Tag
To facilitate the purification of CLuc secreted in a culture supernatant, a plasmid “pCLuRA-TDH3[αP21L,-(GS)3H6]” was constructed, in which a histidine tag was fused to the C-terminus and αCLuc (SEQ ID NO: 20) containing αP21L mutation was expressed. SEQ ID NO: 23 is a partial nucleotide sequence of the plasmid pCLuRA-TDH3[αP21L,-(GS)3H6].
The construction method is as follows.
First, the following PCR was carried out using pCLuRA-TDH3 as a template. Oligo DNA primers used herein are CLuc (GS)3H6-F: CACCACCATCACCACCATTAGTCTAGAGGGCCGCATCATGTAATT (SEQ ID NO: 21) and CLuc (GS)3H6-R: AGAACCAGAACCAGAACCTTTGCATTCATCTGGTACTTCTAGGGT (SEQ ID NO: 22). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; CLuc (GS)3H6-F (SEQ ID NO: 21) (10 pmol/μl): 1.5 μl; CLuc (GS)3H6-R (SEQ ID NO: 22) (10 pmol/μl): 1.5 μl; plasmid pCLuRA-TDH3 solution (10 ng/μl): 0.1 μl; and sterile water: 34 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 48° C. for 30 seconds (annealing), and 68° C. for 8 minutes (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 7.5-kbp DNA fragment was confirmed. This fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation.
Next, both 5′ ends of the thus obtained DNA fragments were phosphorylated using T4 polynucleotide kinase. They were ligated as DNA substrates using T4 DNA ligase for circularization. Escherichia coli DH5α was transformed using the circularized DNA. A plasmid was extracted and purified by a conventional method from the transformed Escherichia coli. The plasmid was subjected to double digestion with EcoR I and Xba I and then the digest was separated by agarose gel electrophoresis. Next, an approximately 1.1-kbp fragment containing a region encoding a histidine tag was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation (DNA fragment G).
Meanwhile, pCLuRA-TDH3[αP21L] was subjected to double digestion with EcoR I and Xba I and then the digest was separated by agarose electrophoresis. An approximately 6.5-kbp fragment was similarly purified (DNA fragment H).
Next, DNA fragment G and DNA fragment H were ligated as DNA substrates using T4 DNA ligase and then Escherichia coli DH5α was transformed with the resultant. The plasmid was extracted and purified by a conventional method from the transformed Escherichia coli. The thus obtained plasmid was subjected to examination of the nucleotide sequence (ranging from positions 1 to positions 2875 of SEQ ID NO: 23), so as to confirm that the sequence was the desired nucleotide sequence. Thus, pCLuRA-TDH3[αP21L,-(GS)3H6] was prepared.
Furthermore, the Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed according to the method described in Example 2 using pCLuRA-TDH3 and pCLuRA-TDH3[αP21L,-(GS)3H6] separately. Wild-type CLuc and CLuc labeled with a histidine tag were each secreted and then each emission spectrum was measured according to the method described in Example 2. As a result, no difference was observed in emission spectrum between the two. Specifically, it was confirmed that no difference in emission spectrum is caused due to the presence or the absence of a histidine tag.
Example 7
A Group of Mutant CLuc, each Having a Substitution of Lysine Corresponding to Amino Acid at Position 375 of SEQ ID NO: 2 with Another Amino Acid
7-1. Plasmid for Expression of a Group of Mutant Cluc Each Having a Substitution of Lysine Corresponding to Amino Acid at Position 375 with Another Amino Acid
As described in Example 2, substitution of lysine corresponding to amino acid at position 375 of SEQ ID NO: 2 with arginine or glutamic acid results in a shift of the emission spectral peak to the longer wavelength side. Hence, a group of plasmids was constructed by the methods described in the following sections 7-2 and 7-3 for secretion and expression of a group of mutant CLuc (and wild-type CLuc) in which amino acid at position 375 was one of 20 types of amino acid composing general proteins.
7-2. Construction 1 of Expression Plasmid
Saturation mutant libraries of amino acid corresponding to amino acid at position 375 of SEQ ID NO: 2 were constructed by 4 types of PCR and intracellular recombination, as follows.
(1) PCR1
Oligo DNA primers used herein are K446X-F: TGAAGTAGAGAAAGTACGAATCAGGNNNCAATCGACTGTAGTAGTAGAACTC A (SEQ ID NO: 24) and mut-CLuc-R (SEQ ID NO: 13). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; K446X-F (SEQ ID NO: 24) (10 pmol/μl): 0.6 μl; mut-CLuc-R (SEQ ID NO: 13) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3[αP21L,-(GS)3H6] solution (1 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles of 94° C. for 15 seconds (denaturation), 45° C. for 30 seconds (annealing), and 68° C. for 1 minute and 30 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 700-bp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution J.”
(2) PCR2
Oligo DNA primers used herein are K446-R: CCTGATTCGTACTTTCTCTACTTCA (SEQ ID NO: 25) and mut-CLuc-F (SEQ ID NO: 8). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; K446-R (SEQ ID NO: 25) (10 pmol/μl): 0.6 μl; mut-CLuc-F (SEQ ID NO: 8) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3[αP21L,-(GS) 3H6] solution (1 ng/μl): 1 μl; and sterile water: 14.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 45° C. for 30 seconds (annealing), and 68° C. for 1 minute and 30 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 1.1-kbp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 10 μl of TE buffer, thereby preparing “DNA solution K.”
(3) PCR3
Oligo DNA primers used herein are mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-R: (SEQ ID NO: 13). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; mut-CLuc-F (SEQ ID NO: 8) (10 pmol/μl): 1.5 μl; mut-CLuc-R (SEQ ID NO: 13) (10 pmol/μl): 1.5 μl; DNA solution J: 1 μl; DNA solution K: 1 μl; and sterile water: 33 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 50° C. for 30 seconds (annealing), and 68° C. for 2 minutes and 20 seconds (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 1.8-kbp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 50 μl of TE buffer, thereby preparing “DNA solution L.”
(4) PCR4
Oligo DNA primers used herein are vec-CLuc-F (SEQ ID NO: 14) and vec-CLuc-R: (SEQ ID NO: 10). The composition of the reaction solution of the PCR is as follows: KOD plus DNA polymerase: 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; vec-CLuc-F (SEQ ID NO: 14) (10 pmol/μl): 1.5 μl; vec-CLuc-R (SEQ ID NO: 10) (10 pmol/μl): 1.5 μl; plasmid pCLuRA-TDH3[αP21L,-(GS)3H6] solution (1 ng/μl): 1 μl; and sterile water: 34 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 50° C. for 30 seconds (annealing), and 68° C. for 7 minutes (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 6-kbp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation, and then dissolved in 50 μl of TE buffer, thereby preparing “DNA solution M.”
Next, the Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed using a mixture of equivalent amounts of DNA solution L and DNA solution M and then caused to form colonies. The thus obtained 96 colonies were cultured separately in a buffered SD-ura liquid medium and then DNA containing the plasmid was extracted and purified from each of the media. Escherichia coli DH5α was transformed with these DNA samples. Plasmid DNAs were extracted and purified from the thus obtained Escherichia coli transformants according to a conventional method and then the nucleotide sequences were examined.
As a result, plasmids were obtained in which codons encoding amino acid corresponding to amino acid at position 375 of SEQ ID NO: 2 were codons encoding the following amino acids: alanine, cysteine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. The plasmids were in turn referred to as “pCLuRA-TDH3[αP21L,K375A,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375C,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375D,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375E,-(GS) 3H6],” “pCLuRA-TDH3[αP21L,K375G,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375I,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375K,-(GS)3H6],” “pCLuRA-TDH3[αP2 μL,K375L,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375M,-(GS) 3H6],” “pCLuRA-TDH3[αP21L,K375N,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375Q,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375R,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375S,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375T,-(GS) 3H6],” “pCLuRA-TDH3[αP21L,K375V,-(GS)3H6],” “pCLuRA-TDH3[αP21L,K375W,-(GS)3H6],” and “pCLuRA-TDH3[αP21L,K375Y,-(GS)3H6],” respectively.
7-3. Construction 2 of Expression Plasmid
pCLuRA-TDH3[αP21L,K375F,-(GS)3H6] that was a plasmid in which the codon encoding the amino acid corresponding to amino acid at position 375 of SEQ ID NO: 2 was a codon encoding phenylalanine was constructed as follows.
PCR was carried out using pCLuRA-TDH3[αP21L,-(GS)3H6] as a template. Oligo DNA primers used herein are K446F: TTTCAATCGACTGTAGTAGAACTCA (SEQ ID NO: 26) and K446-R: CCTGATTCGTACTTTCTCTACTTCA (SEQ ID NO: 25). The composition of the reaction solution for the PCR is as follows: KOD plus DNA polymerase: 0.4 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; K446F (SEQ ID NO: 26) (10 pmol/μl): 0.6 μl; K446-R (SEQ ID NO: 25) (10 pmol/μl): 0.6 μl; plasmid pCLuRA-TDH3[αP21L,-(GS)3H6] solution (1 ng/μl): 1 μl; and sterile water: 12.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 48° C. for 30 seconds (annealing), and 68° C. for 8 minutes (extension).
The total amount of the solution obtained by PCR was electrophoresed with 1% agarose, so that an approximately 7.5-kbp DNA fragment was confirmed. The fragment was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation.
Both 5′ ends of the thus obtained DNA fragments were phosphorylated by T4 polynucleotide kinase. They were ligated as DNA substrates using T4 DNA ligase for circularization. Escherichia coli DH5α was transformed using the circularized DNA. The plasmid was extracted and purified by a conventional method from the transformed Escherichia coli. The plasmid was subjected to double digestion with BamH I and Xba I and then the digest was separated by agarose electrophoresis. Furthermore, an approximately 2.6-kbp fragment containing a region encoding αCLuc in which the codon encoding the amino acid corresponding to amino acid at position 375 of SEQ ID NO: 2 had been altered was purified by the use of GeneElute MINUS EtBr SPIN COLUMNS (Sigma) and ethanol precipitation (DNA fragment N).
Meanwhile, pCLuRA-TDH3[αP21L,-(GS)3H6] was subjected to double digestion with BamH I and Xba I and then the digest was separated by agarose gel electrophoresis. An approximately 5-kbp fragment was similarly purified (DNA fragment P).
Next, DNA fragment N and DNA fragment P were ligated as DNA substrates using T4 DNA ligase. Escherichia coli DH5α was transformed with the resultant. A plasmid was extracted and purified by a conventional method from the thus transformed Escherichia coli. The thus obtained plasmid was subjected to examination of the nucleotide sequence (the sequence ranging from positions 1 to 2875 of SEQ ID NO: 23), so as to confirm that the sequence was the desired nucleotide sequence. Thus, pCLuRA-TDH3[αP21L,K375F,-(GS)3H6] was prepared.
Plasmids (referred to as “pCLuRA-TDH3[αP21L,K375H,-(GS)3H6]” and “pCLuRA-TDH3[αP21L,K375P,-(GS)3H6],” respectively) were constructed, in which the codon encoding the amino acid corresponding to amino acid at position 375 of SEQ ID NO: 2 had been substituted with the codon encoding histidine or proline. The construction method employed for them was the same as that employed for construction of pCLuRA-TDH3[αP21L,K375F,-(GS)3H6], except for the use of different oligo DNA primers for PCR. Oligo DNA primers used for construction of pCLuRA-TDH3[αP21L,K375H,-(GS)3H6] are K446H: CATCAATCGACTGTAGTAGAACTCA (SEQ ID NO: 27) and K446-R (SEQ ID NO: 25). Meanwhile, oligo DNA primers used for construction of pCLuRA-TDH3[αP21L,K375P,-(GS)3H6] are K446P: CCACAATCGACTGTAGTAGAACTCA (SEQ ID NO: 28) and K446-R (SEQ ID NO: 25).
7-4. Emission Spectra of a Group of mutant CLuc Having a Substitution of Lysine Corresponding to Position 375 of SEQ ID NO: 2 with Another Amino Acid
The Saccharomyces cerevisiae BY4743ΔPRB1 strain was transformed with each of 20 types of plasmid obtained in the sections 7-2 and 7-3 above. CLuc secreted by Saccharomyces cerevisiae that had been transformed with the plasmid pCLuRA-TDH3[αP21L,K375K,-(GS)3H6] was wild-type CLuc. CLuc secreted by Saccharomyces cerevisiae that had been transformed with each of 19 types of plasmid other than the plasmid was mutant CLuc. They were each cultured by the method described in Example 2 and then emission spectra were measured using the culture supernatants.
Table 1 shows maximum emission spectral wavelengths of the wild-type CLuc and each mutant CLuc.










TABLE 1








Maximum emission spectral



Mutant CLuc
wavelength (nm)









Wild type
454



K375A
462



K375C
461



K375D
461



K375E
462



K375F
461



K375G
460



K375H
461



K375I
462



K375L
462



K375M
461



K375N
462



K375P
459



K375Q
461



K375R
463



K375S
462



K375T
462



K375V
463



K375W
462



K375Y
457










As shown in Table 1, surprisingly, whereas the maximum emission spectral wavelength was 454 nm in the case of the wild-type CLuc, the maximum emission spectral wavelength was 457 nm or more in all the other cases of mutant CLuc. Specifically, all of the mutant CLucs listed in Table 1 were the 1st mutant luciferases (K375A mutant CLuc, K375C mutant CLuc, K375D mutant CLuc, K375E mutant CLuc, K375F mutant CLuc, K375G mutant CLuc, K375H mutant CLuc, K375I mutant CLuc, K375L mutant CLuc, K375M mutant CLuc, K375N mutant CLuc, K375P mutant CLuc, K375Q mutant CLuc, K375R mutant CLuc, K375S mutant CLuc, K375T mutant CLuc, K375V mutant CLuc, K375W mutant CLuc, and K375Y mutant CLuc).
Example 8
Construction of N404 Saturation Mutant Library and Screening for Mutant CLuc
8-1. Construction of N404 Saturation Mutant Library
A mutant library was constructed in which amino acid at position 404 of the amino acid sequence shown in SEQ ID NO: 2 was substituted any one of the other amino acids.
The nucleotide sequence ranging from positions 1 to 2122 of SEQ ID NO: 7 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment a(475).”
The following oligo DNA primers were used for PCR for amplification of fragment a(475): FAR-F (SEQ ID NO: 16) and N475-rev:ctgagagctgtacgggacgga (SEQ ID NO: 29). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(475) is as follows: KOD plus DNA polymerase (TOYOBO Co., Ltd.): 0.4 μl; pCLuRA-TDH3 plasmid solution (3.8 ng/μl): 1 μl; 10×KOD plus buffer: 2 μl; 2 mM each dNTP mixture: 2 μl; 25 mM magnesium sulfate: 0.8 μl; FAR-F (SEQ ID NO: 16): 0.6 μl (10 pmol/μl); N475-rev (SEQ ID NO: 29): 0.6 μl (10 pmol/μl); and sterile water: 13.6 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 49° C. for 30 seconds (annealing), and 68° C. for 2 minutes and 30 seconds (extension).
Meanwhile, the nucleotide sequence ranging from positions 2102 to 2875 of SEQ ID NO: 7 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment b(475X).”
The following oligo DNA primers were used for PCR for amplification of fragment b(475X): N475X-Fw: tccgtcccgtacagctctcagnnnacttccatctactggcaagat (SEQ ID NO: 30) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(475X) was the same as that employed for amplification of fragment a(475) except for primers. The PCR reaction conditions were the same as those employed for amplification of fragment a(475) except for annealing temperature. The annealing temperature employed herein was 50° C.
The thus obtained PCR products of fragment a(475) and fragment b(475X) were electrophoresed with 1% agarose, so that an approximately 2100-bp fragment a(475) and an approximately 800-bp fragment b(475X) could be confirmed. They were mixed and then subjected to purification using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), phenol extraction, and then ethanol precipitation. The resultant was dissolved in 10 μl of sterile water (the mixed solution of fragments a(475) and b(475X)).
Next, overlap PCR was carried out using the above mixed solution of fragments a(475) and b(475X), as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which the codon at target mutation position was substituted with NNN. Hereinafter, the DNA fragment is referred to as “fragment c(475X).”
The following oligo DNA primers were used for PCR for amplification of fragment c(475X): mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13). Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(475X) is as follows: KOD plus DNA polymerase (TOYOBO Co., Ltd.): 1 μl; a mixed solution of fragments a(475) and b(475X): 1 μl; 10×KOD plus buffer: 5 μl; 2 mM each dNTP mixture: 5 μl; 25 mM magnesium sulfate: 2 μl; mut-CLuc-CF1 (SEQ ID NO: 12): 1.5 μl (10 pmol/μl); mut-CLuc-R (SEQ ID NO: 13): 1.5 μl (10 pmol/μl); and sterile water: 33 μl. PCR was carried out for 1 cycle of 94° C. for 2 minutes (deactivation of anti-polymerase antibody) and 30 cycles each consisting of 94° C. for 15 seconds (denaturation), 61° C. for 30 seconds (annealing), and 68° C. for 1 minute (extension).
Meanwhile, a linear DNA fragment lacking the nucleotide sequence ranging from positions 1664 to 2575 of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d.”
The following oligo DNA primers were used for PCR for amplification of fragment d: SQ-CLuc-CR1 (SEQ ID NO: 15) and vec-CLuc-F (SEQ ID NO: 14). Furthermore, the composition of the reaction solution for PCR for amplification of fragment d was the same as that employed for amplification of fragment c(475X), except for template DNA and oligo DNA primers. The following template DNA was used: pCLuRA-TDH3 plasmid solution (3.8 ng/μl) (11). The PCR reaction conditions were the same as those employed for amplification of fragment c(475X), except for annealing temperature and extension time. The annealing temperature was 58° C. and the extension time was 8 minutes.
The thus obtained PCR products of fragment c(475X) and fragment d were electrophoresed with 0.7% agarose, so that an approximately 11100-bp fragment c(475X) and an approximately 7000-bp fragment d could be confirmed. They were mixed and then subjected to purification using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), phenol extraction, and then ethanol precipitation. The resultant was dissolved in 10 μl of sterile water (a mixed solution of fragments c(475X) and d).
Next, the Saccharomyces cerevisiae BY4743Δprbl strain was transformed by the lithium acetate method using 10 μl of the mixed solution of fragments c(475X) and d. An SD-Ura agar medium was coated with the resultant, followed by approximately 48 hours of incubation at 30° C. Many colonies that had thus appeared were named “N404 saturation mutant libraries.”
8-2. Screening for Luciferase with an Altered (Shifted) Emission Spectrum and an Amino Acid Mutation at Position 404 of SEQ Id No: 2 and Measurement of an Emission Spectrum
Hereafter, clones suspected of having undergone emission spectral shift were selected by the method same as that employed for the sections 2.3 and 2.4 in Example 2 and then the emission spectra were measured.
As shown in Table 2 below, the emission spectral peaks of the thus selected N404G mutant CLuc and N404S mutant CLuc (the 4th mutant luciferases) were both found to be 458 nm. pCLuRA-TDH3 plasmid that had, with respect to the amino acid sequence shown of SEQ ID NO: 2, a substitution (mutation) of asparagine at position 404 with glycine is defined as “pCLuRA-TDH3[N404G].”
Example 9
Construction of T405 Saturation Mutant Library and Screening for Mutant CLuc
A mutant library was constructed, in which amino acid at position 405 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with any one of the other amino acids. The method for construction is similar to that in Example 8.
The nucleotide sequence ranging from positions 1 to 2125 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is hereinafter referred to as “fragment a(476).”
The following oligo DNA primers were used for PCR for amplification of fragment a(476): FAR-F (SEQ ID NO: 16) and T476-rev: gttctgagagctgtacgggac (SEQ ID NO: 31). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(476) was the same as that employed for amplification of fragment a(475) in Example 8, except for primers. The PCR reaction conditions were the same as those employed for amplification of fragment a(475) in Example 8, except for annealing temperature. Annealing was carried out at 59° C.
Meanwhile, the nucleotide sequence ranging from positions 2105 to 2875 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is hereinafter referred to as “fragment b(476X).”
The following oligo DNA primers were used for PCR for amplification of fragment b(476X): T476X-Fw: gtcccgtacagctctcagaacnnntccatctactggcaagatggt (SEQ ID NO: 32) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for amplification of fragment b(476X) was the same as that employed for amplification of fragment b(475X) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8.
Then, a mixed solution of fragments a(476) and b(476X) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(476) and b(476X) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which the codon at target mutation position was substituted with NNN. Hereinafter, the DNA fragment is referred to as “fragment c(476X).”
The composition of the reaction solution for PCR for amplification of fragment c(476X) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(476) and b(476X). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8.
Moreover, a mixed solution of fragments c (476X) and d was prepared in a manner similar to that in Example 8.
Next, clones suspected of having undergone emission spectral shift were selected in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(476X) and d (Example 8). The emission spectra of the thus obtained clones were measured.
As shown in Table 2, the emission spectral peak of the thus selected T405M mutant CLuc (the 5th mutant luciferase) was 457 nm.
Example 10
Construction of S406 Saturation Mutant Library and Screening for Mutant CLuc
A mutant library was constructed, in which amino acid at position 406 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with any one of the other amino acids. The construction method is similar to that in Example 8.
The nucleotide sequence ranging from positions 1 to 2128 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is referred to as “fragment a(477).”
The following oligo DNA primers were used for PCR for amplification of fragment a(477): FAR-F (SEQ ID NO: 16) and S477-rev: agtgttctgagagctgtacgg (SEQ ID NO: 33). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(477) was the same as that employed for amplification of fragment a(475) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8.
Meanwhile, the nucleotide sequence ranging from positions 2108 to 2875 of SEQ ID NO: 7 was amplified by PCR. The fragment is hereinafter referred to as “fragment b(477X).”
The following oligo DNA primers were used for PCR for amplification of fragment b(477X): S477X-Fw: ccgtacagctctcagaacactnnnatctactggcaagatggtgac (SEQ ID NO: 34) and 3′-UTR (SEQ ID NO: 19). The composition of the reaction solution for PCR for amplification of fragment b(477X) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8.
Next, a mixed solution of fragments a(477) and b(477X) was prepared in a manner similar to that in Example 8.
Furthermore, overlap PCR was carried out using the above mixed solution of fragments a(477) and b(477X) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which the codon at target mutation position was substituted with NNN. Hereinafter, the DNA fragment is referred to as “fragment c(477X).”
The composition of the reaction solution for PCR for amplification of fragment c(477X) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(477) and b(477X). The PCR conditions employed herein were the same as those in Example 8.
Furthermore, a mixed solution of fragments c(477X) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, clones suspected of having undergone emission spectral shift were selected in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(477X) and d. The emission spectra of the thus obtained clones were then measured.
As shown in Table 2, the emission spectral peak of the thus selected S406L mutant CLuc (the 6th mutant luciferase) was 460 nm.
Example 11
Construction of I407 Saturation Mutant Library and Screening for Mutant CLuc
A mutant library was constructed, in which amino acid at position 407 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with any one of the other amino acids. The construction method was similar to that in Example 8.
The nucleotide sequence ranging from positions 1 to 2131 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is referred to as “fragment a(478).”
The following oligo DNA primers were used for PCR for amplification of fragment a(478): FAR-F (SEQ ID NO: 16) and I478-rev: ggaagtgttctgagagctgta (SEQ ID NO: 35). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(478) was the same as that in reaction conditions employed for amplification of fragment a(475) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8, except for annealing temperature. Annealing was carried out at 55° C.
Meanwhile, the nucleotide sequence ranging from positions 2111 to 2875 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is referred to as “fragment b(478X).”
The following oligo DNA primers were used for PCR for amplification of fragment b(478X): I478X-Fw: tacagctctcagaacacttccnnntactggcaagatggtgacata (SEQ ID NO: 36) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(478X) was the same as that employed for amplification of fragment b(475X) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8, except for annealing temperature. Annealing was carried out at 58° C.
The thus obtained PCR products of fragment a(478) and fragment b(478X) were electrophoresed with 1% agarose, so that an approximately 2100-bp fragment a(478) and an approximately 800-bp fragment b(478X) could be confirmed. They were mixed and then subjected to purification using Wizard (trademark) SV Gel and PCR Clean-Up system (Promega), phenol extraction, and then ethanol precipitation. The resultant was dissolved in 10 μl of sterile water (a mixed solution of fragments a(478) and b(478X)).
Next, overlap PCR was carried out using the above mixed solution of fragments a(478) and b(478X) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which the codon at target mutation position was substituted with NNN. Hereinafter, the DNA fragment is referred to as “fragment c(478X).”
The composition of the reaction solution for PCR for amplification of fragment c(478X) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: a mixed solution (1 μl) of fragments a(478) and b(478X). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8 except for annealing temperature. Annealing was carried out at 60° C.
The thus obtained PCR product of fragment c(478X) was electrophoresed with 0.7% agarose, so that an approximately 1100-bp fragment c(478X) could be confirmed. This was mixed with fragment d in Example 8. The mixture was then subjected to purification using Wizard (trademark) SV Gel and PCR Clean-Up system (Promega), phenol extraction, and then ethanol precipitation. The resultant was dissolved in 10 μl of sterile water (a mixed solution of fragments c(478X) and d).
Next, clones suspected of having undergone emission spectral shift were selected in a manner similar to that in Example 8 using 10 μl of a mixed solution of fragments c(478X) and d. The emission spectra of the thus obtained clones were measured.
As shown in Table 2, the emission spectral peak of the thus selected I407A mutant CLuc (the 7th mutant luciferase) was 460 nm.
Example 12
T167K/K375R Double Mutant CLuc
DNA encoding double mutant CLuc (the 1st and the 3 mutant luciferases) was prepared as described below, in which amino acid (threonine) at position 167 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with lysine and amino acid (lysine) at position 375 of the same was substituted with arginine.
The DNA fragment containing an amino acid mutation at position 167 and consisting of the nucleotide sequence ranging from positions 1 to 1663 of SEQ ID NO: 7 is hereinafter referred to as “fragment a(238).”
The following oligo DNA primers were used for PCR for amplification of fragment a(238): FAR-F (SEQ ID NO: 16) and SQ-CLuc-CR1 (SEQ ID NO: 15). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(238) was the same as that employed for amplification of fragment a(475) in Example 8 except for the amount of sterile water, template DNA, and primers. The amount of sterile water and template DNA used herein are as follows: sterile water: 12.6 μl and pCLuRA-TDH3[T167K]: 1 μl (4.5 ng/μl). The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8 except for annealing temperature and extension time. Annealing was carried out at 53° C. and extension was carried out for 2 minutes.
Meanwhile, the DNA fragment containing an amino acid mutation at position 375 and consisting of the nucleotide sequence ranging from positions 1554 to 2875 of SEQ ID NO: 7 is hereinafter referred to as “fragment b(446).”
The following oligo DNA primers were used for PCR for amplification of fragment b(446): mut-CLuc-CF1 (SEQ ID NO: 12) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(446) was the same as that employed for amplification of fragment a(238) except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[αP21L,K375R] (1 μl) (2.0 ng/μl). The PCR reaction conditions employed herein were the same as those employed for amplification of fragment a(238).
The thus obtained PCR products of fragment a(238) and fragment b(446) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 1700-bp DNA fragment and an approximately 1300-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments a(238) and b(446) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a(238) and b(446) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(238,446).”
The following oligo DNA primers were used for PCR for amplification of fragment c(238,446): mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-R (SEQ ID NO: 13). Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(238,446) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA and primers. The following template DNA was used: a mixed solution (1 μl) of fragments a(238) and b(446). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8 except for annealing temperature and extension time. Annealing was carried out at 60° C. and extension was carried out for 2 minutes.
Meanwhile, a linear DNA fragment lacking a portion ranging from positions 967 to 2575 of SEQ ID NO: 7 in the pCLuRA-TDH3 sequence was amplified by PCR. Hereinafter, the DNA fragment is referred to as fragment d(238,446).
The following oligo DNA primers were used for PCR for amplification of fragment d(238,446): vec-CLuc-R (SEQ ID NO: 10) and vec-CLuc-F (SEQ ID NO: 14). Furthermore, the composition of the reaction solution for PCR for amplification of fragment d(238,446) was the same as that employed for amplification of fragment d in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment d in Example 8.
The thus obtained PCR products of fragment c(238,446) and fragment d(238,446) were electrophoresed with 0.7% agarose, so that they were confirmed to be an approximately 1700-bp DNA fragment and an approximately 7000-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments c(238,446) and d (238,446) was prepared in a manner similar to that in Example 8.
Next, the Saccharomyces cerevisiae BY4743Δprbl strain was transformed using 10 μl of the mixed solution of fragments c(238,446) and d(238,446) and Frozen-EZ Yeast Transformation IITM (ZYMO RESEARCH).
Furthermore, emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(238,446) and d(238,446).
As shown in Table 2, the emission spectral peak of the thus prepared T167K/K375R double mutant CLuc (the 1st and the 3rd mutant luciferases) was 460 nm. A pCLuRA-TDH3 plasmid having, with respect to the amino acid sequence shown in SEQ ID NO: 2, amino acid at position 167 mutated from threonine to lysine and amino acid at position 375 mutated from lysine to arginine is defined as “pCLuRA-TDH3[T167K,K375R].”
Example 13
T167K/Q403P Double Mutant CLuc
A gene encoding double (T167K and Q403P) mutant CLuc (the 3rd mutant luciferase) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
The DNA fragment containing an amino acid mutation at position 403 and consisting of the nucleotide sequence ranging from positions 1554 to 2875 of SEQ ID NO: 7 is hereinafter referred to as “fragment b(474).”
Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(474) was the same as that employed for amplification of fragment b(446) in Example 12 except for template DNA. The following template DNA was used: pCLuRA-TDH3[Q403P] (pCLuRA-TDH3 plasmid obtained as a result of screening as described in Example 2, in which amino acid at position 403 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from glutamine to proline) (1 μl) (2.56 ng/μl). The PCR conditions were the same as those employed for amplification of fragment b(446) in Example 12.
Next, a mixed solution of fragments a(238) (Example 12) and b(474) was prepared in a manner similar to that in Example 12.
Overlap PCR was carried out using the above mixed solution of fragments a(238) and b(474) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(238,474).”
The composition of the reaction solution for PCR for amplification of fragment c(238,474) was the same as that employed for amplification of fragment c(238,446) in Example 12 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(238) and b(474). The PCR conditions were the same as those in Example 12.
The thus obtained PCR products of fragment c(238,474) and fragment d(238,446) in Example 12 were electrophoresed with 0.7% agarose, so that a mixed solution of fragments c(238,474) and d(238,446) was prepared in a manner similar to that in Example 12.
Next, the emission spectra of the thus obtained clones were measured using 10 μl of the mixed solution of fragments c(238,474) and d(238,446) in a manner similar to that in Example 12.
As shown in Table 2, the emission spectral peak of the thus prepared T167K/Q403P double mutant CLuc (the 3rd mutant luciferase) was 458 nm.
Example 14
T167K/N404G Double Mutant CLuc
A gene encoding double (T167K and N404G) mutant CLuc (the 3rd and the 4th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment containing an amino acid mutation at position 404 and consisting of the nucleotide sequence ranging from positions 1554 to 2875 of SEQ ID NO: 7 is hereinafter referred to as “fragment b(475).”
The composition of the reaction solution for PCR for amplification of fragment b(475) was the same as that employed for amplification of fragment b(446) in Example 12 except for template DNA. The following template DNA was used: pCLuRA-TDH3[N404G] (Example 8) (1 μl) (2.70 ng/μl). The PCR conditions were the same as those employed for amplification of fragment b(446) in Example 12.
Next, a mixed solution of fragments a(238) (Example 12) and b(475) was prepared in a manner similar to that in Example 12.
Overlap PCR was carried out using the above mixed solution of fragments a(238) and b(475) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(238,475).”
The composition of the reaction solution for PCR for amplification of fragment c(238,475) was the same as that employed for amplification of fragment c(238,446) in Example 12 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(238) and b(475). The PCR conditions were the same as those in Example 12.
The thus obtained PCR products of fragment c(238,475) and fragment d(238,446) in Example 12 were electrophoresed with 0.7% agarose, so that a mixed solution of fragments c(238,475) and d(238,446) was prepared in a manner similar to that in Example 12.
Next, the emission spectra of the thus obtained clones were measured using 10 μl of the mixed solution of fragments c(238,475) and d(238,446) in a manner similar to that in Example 12.
As shown in Table 2, the emission spectral peak of the thus prepared T167K/N404G double mutant CLuc (the 3rd and the 4th mutant luciferases) was 460 nm.
Example 15
T167K/T405I Double Mutant CLuc
A gene encoding a double (T167K and T405I) mutant CLuc (the 3rd and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment containing an amino acid mutation at position 405 and consisting of the nucleotide sequence ranging from positions 1554 to 2875 of SEQ ID NO: 7 is hereinafter referred to as “fragment b(476).”
The composition of the reaction solution for PCR for amplification of fragment b(476) was the same as that employed for amplification of fragment b(446) in Example 12 except for template DNA. The following template DNA was used: pCLuRA-TDH3[αP21L,T405I] (Example 5) (1 μl) (2.0 ng/μl). The PCR conditions were the same as those employed for amplification of fragment b(446) in Example 12.
Next, a mixed solution of fragments a(238) (Example 12) and b(476) was prepared in a manner similar to that in Example 12.
Overlap PCR was carried out using the above mixed solution of fragments a(238) and b(476) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(238,476).”
The composition of the reaction solution for PCR for amplification of fragment c(238,476) was the same as that employed for amplification of fragment c(238,446) in Example 12 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(238) and b(476). The PCR conditions were the same as those in Example 12.
The thus obtained PCR products of fragment c(238,476) and fragment d(238,446) in Example 12 were electrophoresed with 0.7% agarose, so that the mixed solution of fragments c(238,476) and d(238,446) was prepared in a manner similar to that in Example 12.
Next, the emission spectra of the thus obtained clones were measured using 10 μl of the mixed solution of fragments c(238,476) and d(238,446) in a manner similar to that in Example 12.
As shown in Table 2, the emission spectral peak of the thus prepared T167K/T405I double mutant CLuc (the 3rd and the 5th mutant luciferases) was 460 nm.
Example 16
Construction of L197 Saturation Mutant Library and Screening for Mutant CLuc
16-1. Construction of L197 Saturation Mutant Library
A mutant library was constructed, in which amino acid at position 197 of the amino acid sequence shown in SEQ ID NO: 2 was substituted with any one of the other amino acids. The construction method is similar to that in Example 8.
The nucleotide sequence ranging from positions 1 to 1501 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is referred to as “fragment a(268).”
The following oligo DNA primers were used for PCR for amplification of fragment a(268): FAR-F (SEQ ID NO: 16) and L268-rev: gatgtcgatcacgatcagttt (SEQ ID NO: 37). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(268) was the same as that employed for amplification of fragment a(475) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8.
Meanwhile, the nucleotide sequence ranging from positions 1481 to 2875 of SEQ ID NO: 7 was amplified by PCR. The DNA fragment is referred to as “fragment b(268X).”
The following oligo DNA primers were used for PCR for amplification of fragment b(268X): L268X-Fw: aaactgatcgtgatcgacatcnnnggaggaagatctgtaagaatc (SEQ ID NO: 38) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(268X) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8.
The thus obtained PCR products of fragment a(268) and fragment b(268X) were electrophoresed with 1% agarose, so that an approximately 1500-bp fragment a(268) and an approximately 1400-bp fragment b(268X) could be confirmed. Next, a mixed solution of fragments a(268) and b(268X) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(268) and b(268X) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 1813 of SEQ ID NO: 7) was prepared, in which the codon at target mutation position was substituted with NNN. Hereinafter, the DNA fragment is referred to as “fragment c(268X).”
The following oligo DNA primers were used for PCR for amplification of fragment c(268X): mut-CLuc-F (SEQ ID NO: 8) and mut-CLuc-NR2 (SEQ ID NO: 9). Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(268X) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA and primers. The following template DNA was used: the mixed solution (1 μl) of fragments a(268) and b(268X). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8 except for annealing temperature. Annealing was carried out at 53° C.
Meanwhile, linear DNA lacking a portion ranging from positions 967 to 1703 of SEQ ID NO: 7 in the pCLuRA-TDH3 sequence was amplified by PCR. The DNA fragment is referred to as “fragment d(268).”
The following oligo DNA primers were used for PCR for amplification of fragment d(268): vec-CLuc-R (SEQ ID NO: 10) and SQ-CLuc-NF2 (SEQ ID NO: 11). Furthermore, the composition of the reaction solution for PCR for amplification of fragment d(268) was the same as that employed for amplification of fragment d in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment d in Example 8 except for annealing temperature. Annealing was carried out at 62° C.
The thus obtained PCR products of fragment c(268X) and fragment d(268) were electrophoresed with 0.7% agarose, so that an approximately 900-bp fragment c(268X) and an approximately 7000-bp fragment d(268) could be confirmed.
Next, a mixed solution of fragments c(268X) and d(268) was prepared in a manner similar to that in Example 8.
Furthermore, in a manner similar to that in Example 8 using the mixed solution of fragments c(268X) and d(268), a clone with an altered spectral peak position was obtained, in which amino acid at position 197 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from leucine to proline. A pCLuRA-TDH3 plasmid, in which amino acid at position 197 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from leucine to proline, is defined as “pCLuRA-TDH3[L197P].”
16-2. Preparation of M178K/L197P Double Mutant CLuc Gene
A gene encoding double (M178K and L197P) mutant CLuc (the 2nd mutant luciferase) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment containing amino acid mutation at position 178 shown in SEQ ID NO: 2 and consisting of the nucleotide sequence ranging from positions 1 to 1492 of SEQ ID NO: 7 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment a(249).”
The following oligo DNA primers were used for PCR for amplification of fragment a(249): FAR-F (SEQ ID NO: 16) and SQ-CLuc-F001-rev:cacgatcagtttgaagaattctatgacggt (SEQ ID NO: 39). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(249) was the same as that for amplification of fragment a(475) in Example 8, except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[M178K] (Example 2) (1 μl) (2.85 ng/μl). The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8, except for annealing temperature. Annealing was carried out at 58° C.
Meanwhile, a DNA fragment containing an amino acid mutation at position 197 of SEQ ID NO: 2 and consisting of the nucleotide sequence ranging from positions 1463 to 2875 of SEQ ID NO: 7 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment b(268).”
The following oligo DNA primers were used for PCR for amplification of fragment b(268): mut-CLuc-CF0: accgtcatagaattcttcaaactgatcgtg (SEQ ID NO: 40) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(268) was the same as that for amplification of fragment a(249), except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[L197P] (the section 16-1 above) (1 μl) (3.53 ng/μl). The PCR conditions were the same as those employed for amplification of fragment a(249).
The thus obtained PCR products of fragment a(249) and fragment b(268) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 1500-bp DNA fragment and an approximately 1400-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments a(249) and b(268) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a(249) and b(268) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 900 to 1813 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(249,268).”
Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(249,268) was the same as that employed for amplification of fragment c(268X) in the section 16-1 above except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(249) and b(268). The PCR conditions were the same as those employed for amplification of fragment c(268X) in the section 16-1 above, except for annealing temperature and extension time alone. Annealing was carried out at 60° C. and extension was carried out for 1 minute and 30 seconds.
The thus obtained PCR products of fragment c(249,268) and fragment d(268) in the section 16-1 above were electrophoresed with 0.7% agarose, so that they were confirmed to be an approximately 800-bp DNA fragment and an approximately 7000-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments c(249,268) and d(268) was prepared in a manner similar to that in Example 8.
Next, in a manner similar to that in Example 8, a clone was obtained, in which, amino acid at position 178 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from methionine to lysine and amino acid at position 197 of the same was mutated from leucine to proline. A pCLuRA-TDH3 plasmid, in which, amino acid at position 178 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from methionine to lysine and amino acid at position 197 of the same was mutated from leucine to proline is defined as “pCLuRA-TDH3[M178K,L197P].”
16-3. Mutagenesis of Amino Acid at Position 21 within the Signal Sequence of αCLuc
Amino acid at position 21 within the signal sequence of αCLuc shown in SEQ ID NO: 6 was substituted with leucine. Mutagenesis is carried out as follows. A DNA fragment containing a mutation corresponding to position 21 in the amino acid sequence shown in SEQ ID NO: 6 and a DNA fragment containing two mutations at positions 178 and 197 of the amino acid sequence shown in SEQ ID NO: 2 were amplified by PCR. Preparation was carried out by overlap PCR using the two DNA fragments.
A DNA fragment consisting of the nucleotide sequence ranging from positions 1 to 966 of SEQ ID NO: 7 and containing an amino acid mutation at position 21 shown in SEQ ID NO: 6 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment a(21).”
The composition of the reaction solution for PCR for amplification of fragment a(21) was the same as that employed for amplification of fragment a(238) in Example 12 except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L] (1 μl) (4.25 ng/μl), and FAR-F (SEQ ID NO: 16) and vec-CLuc-R (SEQ ID NO: 10). The PCR conditions were the same as those employed for amplification of fragment a(238) in Example 12, except for annealing temperature. Annealing was carried out at 53° C.
Meanwhile, a DNA fragment consisting of the nucleotide sequence ranging from positions 900 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 178 and 197 of SEQ ID NO: 2 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment b(249,268).”
The composition of the reaction solution for PCR for amplification of fragment b(249,268) was the same as that employed for amplification of fragment a(21) except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[M178K,L197P] (the section 16-2 above) (1 μl) (1.56 ng/μl), and mut-CLuc-F (SEQ ID NO: 8) and 3′-UTR (SEQ ID NO: 19). The PCR conditions were the same as those employed for amplification of fragment a(21).
The thus obtained PCR products of fragment a(21) and fragment b(249,268) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 1000-bp DNA fragment and an approximately 1900-bp DNA fragment. They were mixed and then a mixed solution of fragments a(21) and b(249,268) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a(21) and b(249,268) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 461 to 1813 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(21,249,268).”
The following oligo DNA primers were used for PCR for amplification of fragment c(21,249,268): SQ-GPD1-F0:ATGTATCTATCTCATTTTCTTACA (SEQ ID NO: 41) and mut-CLuc-NR2 (SEQ ID NO: 9). Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(21,249,268) was the same as that employed for amplification of fragment c(238,446) in Example 12, except for template DNA and primers. The following template DNA was used: the mixed solution (1 μl) of fragments a(21) and b(249,268). The PCR conditions were the same as those employed for amplification of fragment a(238) in Example 12, except for annealing temperature. Annealing was carried out at 51° C.
Meanwhile, a linear DNA fragment lacking a portion ranging from positions 526 to 1703 of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d(21,249,268).”
The following oligo DNA primers were used for PCR for amplification of fragment d(21,249,268): SQ-GPD 1-R0:CAGCTTTTTCCAAATCAGAGAGAGCAG (SEQ ID NO: 42) and SQ-CLuc-NF2 (SEQ ID NO: 11). Furthermore, the composition of the reaction solution for PCR for amplification of fragment d(21,249,268) was the same as that employed for amplification of fragment d(238,446) in Example 12 except for primers. The PCR conditions were the same as those employed for amplification of fragment d(238,446) in Example 12.
The thus obtained PCR products of fragments c(21,249,268) and d(21,249,268) were electrophoresed with 0.7% agarose, so that they were confirmed to be an approximately 1300-bp DNA fragment and an approximately 7000-bp DNA fragment. They were mixed and then a mixed solution of fragments c(21,249,268) and d(21,249,268) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using mixed solution of fragments c(21,249,268) and d(21,249,268).
As shown in Table 2, the emission spectral peak of the thus prepared M178K/L197P double mutant CLuc (the 2nd mutant luciferase) was 447 nm.
Example 17
K375R/Q403P Double Mutant CLuc
A gene encoding double (K375R and Q403P) mutant CLuc (the 1st mutant luciferase) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 900 to 2087 of SEQ ID NO: 7 and containing an amino acid mutation at position 375 is hereinafter referred to as “fragment a(446).”
The following oligo DNA primers were used for PCR for amplification of fragment a(446): mut-CLuc-F (SEQ ID NO: 8) and SQ-CLuc-F002-rev: caaccagaatctgttttccatcaa (SEQ ID NO: 43). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(446) was the same as that employed for amplification of fragment a(238) in Example 12, except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[αP21L,K375R] (Example 4) (1 μl) (2.0 ng/μl). The PCR conditions were the same as those employed for amplification of fragment a(238) in Example 12.
Meanwhile, a DNA fragment consisting of nucleotide sequence ranging from positions 2064 to 2875 of SEQ ID NO: 7 and containing an amino acid mutation at position 403 is hereinafter referred to as “fragment b′(474).”
The following oligo DNA primers were used for PCR for amplification of fragment b′(474): SQ-CLuc-F002: ttgatggaaaacagattctggttg (SEQ ID NO: 44) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b′(474) was the same as that employed for amplification of fragment b(474) in Example 13 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(446) in Example 12.
The thus obtained PCR products of fragments a(446) and b′(474) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 1100-bp DNA fragment and an approximately 800-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments a(446) and b′(474) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a(446) and b′(474) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(446,474).”
The composition of the reaction solution for PCR for amplification of fragment c(446,474) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a (446) and b′(474). The PCR conditions were the same as those employed for amplification of fragment c(238,446) in Example 12.
The thus obtained PCR product of fragment c(446,474) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(446,474) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 12 using 10 μl of the mixed solution of fragments c(446,474) and d.
As shown in Table 2, the emission spectral peak of the thus prepared K375R/Q403P double mutant CLuc (the 1st mutant luciferase) was 460 nm.
Example 18
K375R/N404G Double Mutant CLuc
A gene encoding double (K375R and N404G) mutant CLuc (the 1st and the 4th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2064 to 2875 of SEQ ID NO: 7 and containing an amino acid mutation at position 404 is hereinafter referred to as “fragment b′(475).”
The composition of the reaction solution for PCR for amplification of fragment b′(475) was the same as that employed for amplification of fragment b′(474) in Example 17 except for template DNA. The following template DNA was used: pCLuRA-TDH3[N404G] (Example 8) (1 μl) (2.70 ng/μl). The PCR conditions were the same as those in Example 12.
The thus obtained PCR products of fragments a(446) and b′(475) in Example 17 were electrophoresed with 1% agarose. A mixed solution of fragments a(446) and b′(475) was prepared in a manner similar to that in Example 12.
Next, overlap PCR was carried out using the above mixed solution of fragments a(446) and b′(475) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(446,475).”
The composition of the reaction solution for PCR for amplification of fragment c(446,475) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(446) and b′(475). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8.
The thus obtained PCR product of fragment c(446,475) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(446,475) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 12 using 10 μl of the mixed solution of fragments c(446,475) and d.
As shown in Table 2, the emission spectral peak of the thus prepared K375R/N404G double mutant CLuc (the 1st and the 4 h mutant luciferases) was 461 nm.
Example 19
K375R/T405I Double Mutant CLuc
A gene encoding double (K375R and T405I) mutant CLuc (the 1st and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2064 to 2875 of SEQ ID NO: 7 and containing an amino acid mutation at position 405 is hereinafter referred to as “fragment b′(476).”
The composition of the reaction solution for PCR for amplification of fragment b′(476) was the same as that employed for amplification of fragment b′(474) in Example 17, except for template DNA. The following template DNA was used: pCLuRA-TDH3[αP21L,T405I] (Example 5) (1 μl) (2.0 ng/11). The PCR conditions were the same as those employed for amplification of fragment b(446) in Example 12.
The thus obtained PCR products of fragment a(446) of Example 17 and fragment b′(476) were electrophoresed with 1% agarose. A mixed solution of fragments a(446) and b′(476) was prepared in a manner similar to that in Example 17.
Next, overlap PCR was carried out using the above mixed solution of fragments a(446) and b′(476) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(446,476).”
The composition of the reaction solution for PCR for amplification of fragment c(446,476) was the same as that employed for PCR in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(446) and b′(476). The PCR conditions were the same as those employed for amplification of fragment c(238,446) in Example 12.
The thus obtained PCR product of fragment c(446,476) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(446,476) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(446,476) and d.
As shown in Table 2, the emission spectral peak of the thus prepared K375R/T405I double mutant CLuc (the 1st and the 5th mutant luciferases) was 463 nm.
Example 20
Q403P/N404G Double Mutant CLuc
A gene encoding double (Q403P and N404G) mutant CLuc (the 4th mutant luciferase) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 1 to 2119 of SEQ ID NO: 7 is hereinafter referred to as “fragment a(474).”
The following oligo DNA primers were used for PCR for amplification of fragment a(474): FAR-F (SEQ ID NO: 16) and Q474-rev:agagctgtacgggacggacac (SEQ ID NO: 45). Furthermore, the composition of the reaction solution for PCR for amplification of fragment a(474) was the same as that employed for amplification of fragment a(475) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment a(475) in Example 8, except for annealing temperature. Annealing was carried out at 55° C.
Meanwhile, a DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403 and 404 is hereinafter referred to as “fragment b(474,475).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,475): Q474P/N475G-Fw:gtgtccgtcccgtacagctctcccgggacttccatctactggcaagat (SEQ ID NO: 46) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(474,475) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8, except for annealing temperature. Annealing was carried out at 58° C.
Next, a mixed solution of fragments a(474) and b(474,475) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) and b(474,475) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,475).”
The composition of the reaction solution for PCR for amplification of fragment c(474,475) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,475). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8, except for annealing temperature and extension time. Annealing was carried out at 60° C. and extension was carried out for 1 minute and 30 seconds.
The thus obtained PCR product of fragment c(474,475) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(474,475) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(474,475) and d.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/N404G double mutant CLuc (the 4th mutant luciferase) was 462 nm.
Example 21
Q403P/T405I Double Mutant CLuc
A gene encoding double (Q403P and T405I) mutant CLuc (the 5th mutant luciferase) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutation at position 403 and the same at position 405 is hereinafter referred to as “fragment b(474,476).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,476): Q474P/T476I-Fw: gtgtccgtcccgtacagctctcccaacatctccatctactggcaagatggt (SEQ ID NO: 47) and 3′-UTR (SEQ ID NO: 19). The composition of the reaction solution in PCR for amplification of fragment b(474,476) was the same as that employed for amplification of fragment b(475X) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment b(474,475) in Example 20.
Next, a mixed solution of fragments a(474) and b(474,476) was prepared in a manner similar that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) (Example 20) and b(474,476) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,476).”
The composition of the reaction solution in PCR for amplification of fragment c(474,476) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,476). The PCR conditions were the same as those employed for amplification of fragment c(474,475) in Example 20.
The thus obtained PCR product of fragment c(474,476) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(474,476) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured using 10 μl of the mixed solution of fragments c(474,476) and d in a manner similar to that in Example 8.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/T405I double mutant CLuc (the 5th mutant luciferase) was 459 nm.
Example 22
N404G/T405I Double Mutant CLuc
A gene encoding double (N404G and T405I) mutant CLuc (the 4th and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2102 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 404 and 405 is hereinafter referred to as “fragment b(475,476).”
The following oligo DNA primers were used for PCR for amplification of fragment b(475,476): N475G/T476I-Fw: tccgtcccgtacagctctcaggggatctccatctactggcaagatggt (SEQ ID NO: 48) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(475,476) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(474,475) in Example 20.
Next, a mixed solution of fragments a(475) (Example 8) and b(475,476) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(475) and b(475,476) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(475,476).”
The composition of the reaction solution in PCR for amplification of fragment c(475,476) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(475) and b(475,476). The PCR conditions were the same as those employed for amplification of fragment c(474,475) in Example 20.
The thus obtained PCR product of fragment c(475,476) was electrophoresed with 0.7% agarose. The experiment was conducted in a manner similar to that in Example 8, so that a mixed solution of fragments c(475,476) and d (Example 8) was prepared.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(475,476) and d.
As shown in Table 2, the emission spectral peak of the thus prepared N404G/T405I double mutant CLuc (the 4th and the 5th mutant luciferases) was 461 nm.
Example 23
Q403P/N404G/T405I Triple Mutant CLuc and Q403P/N404G/T405M Triple Mutant CLuc
23-1. Preparation (1) of Triple Mutant CLuc Gene with Mutations at Positions 403, 404, and 405
A gene encoding triple (Q403P, N404G, and T405I) mutant CLuc (the 4th and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, and 405 is hereinafter referred to as “fragment b(474,475,476I).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,475,476I): Q474P/N475G/T476I-Fw: gtgtccgtcccgtacagctctcccgggatctccatctactggcaagatggt (SEQ ID NO: 49) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution in PCR for amplification of fragment b(474,475,476I) was the same as that employed for amplification of fragment b(475X) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment b(474,475) in Example 20.
Next, a mixed solution of fragments a(474) (Example 20) and b(474,475,476I) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) and b(474,475,476I) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,475,476I).”
The composition of the reaction solution for PCR for amplification of fragment c(474,475,476I) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,475,476I). The PCR conditions were the same as those employed for amplification of fragment c(474,475) in Example 20.
The thus obtained PCR product of fragment c(474,475,476I) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(474,475,476I) and d (Example 8) was prepared in a manner similar to that in Example 8.
The emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(474,475,476I) and d.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/N404G/T405I triple mutant CLuc (the 4th and the 5th mutant luciferases) was 462 nm.
23-2. Preparation (2) of Triple Mutant CLuc Gene with Mutations at Positions 403, 404, and 405
A gene encoding triple (Q403P, N404G, and T405M) mutant CLuc (the 4th and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, and 405 is hereinafter referred to as “fragment b(474,475,476M).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,475,476M): Q474P/N475G/T476M-Fw: gtgtccgtcccgtacagctctcccgggatgtccatctactggcaagatggt (SEQ ID NO: 50) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution in PCR for amplification of fragment b(474,475,476M) was the same as that employed for amplification of fragment b(475X) in Example 8, except for primers. The PCR conditions were the same as those employed for amplification of fragment b(474,475) in Example 20.
Next, a mixed solution of fragments a(474) (Example 20) and b(474,475,476M) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) and b(474,475,476M) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,475,476M).”
The composition of the reaction solution for PCR for amplification of fragment c(474,475,476M) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,475,476M). The PCR conditions employed herein were the same as those employed for amplification of fragment c(474,475) in Example 20.
The thus obtained PCR product of fragment c(474,475,476M) was electrophoresed with 0.7% agarose. A mixed solution of fragments c(474,475,476M) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(474,475,476M) and d.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/N404G/T405M triple mutant CLuc (the 4th and the 5th mutant luciferases) was 462 nm. A pCLuRA-TDH3 plasmid, in which amino acid at position 403 of the amino acid sequence shown in SEQ ID NO: 2 was mutated from glutamine to proline, amino acid at position 404 of the same was mutated from asparagine to glycine, and amino acid at position 405 of the same was mutated from threonine to methionine, is defined as “pCLuRA-TDH3[Q403P,N404G,T405M].”
Example 24
Q403P/N404G/T405M/S406L Quadruple Mutant CLuc
A gene encoding quadruple (Q403P, N404G, T405M, and S406L) mutant CLuc (the 4th to the 6th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, 405, and 406 is hereinafter referred to as “fragment b(474,475,476,477).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,475,476,477): Q474P/N475G/T476M/S477L-Fw: gtgtccgtcccgtacagctctcccgggatgctcatctactggcaagatggtgac (SEQ ID NO: 51) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(474,475,476,477) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(475X) in Example 8 except for annealing temperature. Annealing was carried out at 55° C.
Next, a mixed solution of fragments a(474) (Example 20) and b(474,475,476,477) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) and b(474,475,476,477) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,475,476,477).”
The composition of the reaction solution for PCR for amplification of fragment c(474,475,476,477) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,475,476,477). The PCR conditions were the same as those employed for amplification of fragment c(475X) in Example 8 except for annealing temperature. Annealing was carried out at 61° C.
The thus obtained PCR product of fragment c(474,475,476,477) was electrophoresed with 0.7% agarose and then a mixed solution of fragments c(474,475,476,477) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(474,475,476,477) and d.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/N404G/T405M/S406L quadruple mutant CLuc (the 4th to the 6th mutant luciferases) was 461 nm.
Example 25
Preparation of Q403P/N404G/T405M/S406L/I407A Quintuple Mutant CLuc Gene
A gene encoding quintuple (Q403P, N404G, T405M, S406L, and I407A) mutant CLuc (the 4th to the 7th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 2099 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, 405, 406, and 407 is hereinafter referred to as “fragment b(474,475,476,477,478).”
The following oligo DNA primers were used for PCR for amplification of fragment b(474,475,476,477,478): Q474P/N475G/T476M/S477L/I478A-Fw: gtgtccgtcccgtacagctctcccgggatgctcgcctactggcaagatggtgacata (SEQ ID NO: 52) and 3′-UTR (SEQ ID NO: 19). Furthermore, the composition of the reaction solution for PCR for amplification of fragment b(474,475,476,477,478) was the same as that employed for amplification of fragment b(475X) in Example 8 except for primers. The PCR conditions were the same as those employed for amplification of fragment b(268) in the section 16-2 of Example 16.
Next, a mixed solution of fragments a(474) and b(474,475,476,477,478) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(474) and b(474,475,476,477,478) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 1554 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment c(474,475,476,477,478) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(474) and b(474,475,476,477,478). The PCR conditions were the same as those employed for amplification of fragment c(249,268) in the section 16-2 of Example 16.
The PCR product of fragment c(474,475,476,477,478) was electrophoresed with 0.7% agarose and then a mixed solution of fragments c(474,475,476,477,478) and d (Example 8) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(474,475,476,477,478) and d.
As shown in Table 2, the emission spectral peak of the thus prepared Q403P/N404G/T405M/S406L/I407A quintuple mutant CLuc (the 4th to the 7th mutant luciferases) was 460 nm. A pCLuRA-TDH3 plasmid having, with respect to the amino acid sequence shown in SEQ ID NO: 2, amino acid at position 403 mutated from glutamine to proline, amino acid at position 404 mutated from asparagine to glycine, amino acid at position 405 mutated from threonine to methionine, amino acid at position 406 mutated from serine to leucine and amino acid at position 407 mutated from isoleucine to alanine, is defined as “pCLuRA-TDH3[Q403P,N404G,T405M,S406L,I407A].”
Example 26
Preparation of T167K/Q403P/N404G/T405M/S406L/I407A Sextuple Mutant CLuc Gene
A gene encoding sextuple (T167K, Q403P, N404G, T405M, S406L, and I407A) mutant CLuc (the 3rd to the 7th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Mutagenesis was carried out in a manner similar to that in Example 12.
Meanwhile, a DNA fragment consisting of the nucleotide sequence ranging from positions 1 to 1813 of SEQ ID NO: 7 and containing an amino acid mutation at position 167 is hereinafter referred to as “fragment a′(238).”
The composition of the reaction solution for PCR for amplification of fragment a′(238) was the same as that employed for amplification of fragment a(238) in Example 12 except for primers. The following oligo DNA primers were used: FAR-F (SEQ ID NO: 16) and mut-CLuc-NR2 (SEQ ID NO: 9). The PCR conditions employed herein were the same as those employed for amplification of fragment a(475) in Example 8, except for annealing temperature. Annealing was carried out at 53° C.
Meanwhile, a DNA fragment consisting of the nucleotide sequence ranging from positions 1704 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, 405, 406, and 407 is hereinafter referred to as “fragment b′(474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment b′(474,475,476,477,478) was the same as that employed for amplification of fragment b(446) in Example 12 except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[Q403P,N404G,T405M,S406L,I407A] (Example 25) (1 μl) (1.45 ng/μl), SQ-CLuc-NF2 (SEQ ID NO: 11), and 3′-UTR (SEQ ID NO: 19). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment a′(238).
The PCR products of the thus obtained fragments a′(238) and b′(474,475,476,477,478) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 1800-bp DNA fragment and an approximately 1200-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments a′(238) and b′(474,475,476,477,478) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a′(238) and b′(474,475,476,477,478) as a template. Thus, one long fragment (the nucleotide sequence ranging from positions 461 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(238,474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment c(238,474,475,476,477,478) was the same as that employed for amplification of fragment c(475X) in Example 8 except for template DNA and primers. The following template DNA and oligo DNA primers were used: the mixed solution (1 μl) of fragments a′(238) and b′(474,475,476,477,478), SQ-GPD1-F0 (SEQ ID NO: 41), and mut-CLuc-R (SEQ ID NO: 13). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment c(475X) in Example 8, except for annealing temperature and extension time. Annealing was carried out at 51° C. and extension was carried out for 2 minutes and 30 seconds.
Meanwhile, a linear DNA fragment lacking a portion ranging from positions 526 to 2575 of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d(238,474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment d(238,474,475,476,477,478) was the same as that employed for amplification of fragment d in Example 8 except for primers. The following oligo DNA primers were used in the PCR: SQ-GPD1-R0 (SEQ ID NO: 42) and vec-CLuc-F (SEQ ID NO: 14). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment d in Example 8.
The PCR products of fragments c(238,474,475,476,477,478) and d(238,474,475,476,477,478), were electrophoresed with 0.7% agarose, so that they were confirmed to be an approximately 2200-bp DNA fragment and an approximately 7000-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments c(238,474,475,476,477,478) and d(238,474,475,476,477,478) was prepared in a manner similar to that in Example 8.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(238,474,475,476,477,478) and d(238,474,475,476,477,478).
As shown in Table 2, the emission spectral peak of the thus prepared T167K/Q403P/N404G/T405M/S406L/I407A sextuple mutant CLuc (the 3rd to the 7th mutant luciferases) was 461 nm.
Example 27
K375R/Q403P/N404G/T405M/S406L/I407A Sextuple Mutant CLuc
A gene encoding sextuple (K375R, Q403P, N404G, T405M, S406L, and I407A) mutant CLuc (the 1st and the 4th to the 7th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Furthermore, amino acid at position 21 within the signal sequence of αCLuc shown in SEQ ID NO: 6 was substituted with leucine. Mutagenesis was carried out in a manner similar to that in Example 12.
The DNA fragment consisting of the nucleotide sequence ranging from positions 1 to 2087 of SEQ ID NO: 7 and containing an amino acid mutation at position 21 of SEQ ID NO: 6 and an amino acid mutation at position 375 of SEQ ID NO: 2 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment a(21,446).”
The composition of the reaction solution for PCR for amplification of fragment a(21,446) was the same as that employed for amplification of fragment a(238) in Example 12 except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L,K375R] (Example 4) (1 μl) (2.0 ng/μl), FAR-F (SEQ ID NO: 16), and SQ-CLuc-F002-rev (SEQ ID NO: 43). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment a(21) in the section 16-3 of Example 16.
Meanwhile, a DNA fragment consisting of the nucleotide sequence ranging from positions 2064 to 2875 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, 405, 406, and 407 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment b′′(474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment b′′(474,475,476,477,478) was the same as that employed for amplification of fragment a(238) in Example 12 except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[Q403P,N404G,T405M,S406L,I407A] (Example 25) (1 μl) (1.45 ng/μl), SQ-CLuc-F002 (SEQ ID NO: 44), and 3′-UTR (SEQ ID NO: 19). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment a(21) in the section 16-3 of Example 16.
The PCR products of the thus obtained fragment a(21,446) and fragment b′′(474,475,476,477,478) were electrophoresed with 1% agarose, so that they were confirmed to be an approximately 2100-bp DNA fragment and an approximately 800-bp DNA fragment, respectively. They were mixed, so that a mixed solution of fragments a(21,446) and b′′(474,475,476,477,478) was prepared in a manner similar to that in Example 8.
Next, overlap PCR was carried out using the above mixed solution of fragments a(21,446) and b′′(474,475,476,477,478) as a template. Thus, one long fragment (comprising the nucleotide sequence ranging from positions 461 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(21,446,474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment c(21,446,474,475,476,477,478) was the same as that employed for amplification of fragment c(238,474,475,476,477,478) in Example 26 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(21,446) and b′′(474,475,476,477,478). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment c(21,249,268) in the section 16-3 of Example 16.
The thus obtained PCR product of fragment c(21,446,474,475,476,477,478) was electrophoresed with 0.7% agarose, so that a mixed solution of fragments c(21,446,474,475,476,477,478) and d(238,474,475,476,477,478) (Example 26) was prepared in a manner similar to that in Example 26.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(21,446,474,475,476,477,478) and d(238,474,475,476,477,478).
As shown in Table 2, the emission spectral peak of the thus prepared K375R/Q403P/N404G/T405M/S406L/I407A sextuple mutant CLuc (the 1st and the 4th to the 7th mutant luciferases) was 460 nm.
Example 28
T167K/K375R/Q403P/N404G/T405M/S406L/I407A Septuple Mutant CLuc
A gene encoding septuple (T167K, K375R, Q403P, N404G, T405M, S406L, and I407A) mutant CLuc (the 1st and the 3rd to the 7th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2 was prepared. Furthermore, amino acid at position 21 within the signal sequence of αCLuc shown in SEQ ID NO: 6 was substituted with leucine. Mutagenesis was carried out in a manner similar to that in Example 12.
A DNA fragment consisting of the nucleotide sequence ranging from positions 900 to 2087 of SEQ ID NO: 7 and containing mutations at amino acid positions 167 and 375 was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment a′(238,446).”
The composition of the reaction solution for PCR for amplification of fragment a′(238,446) was the same as that employed for amplification of fragment a(238) in Example 12 except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[T167K,K375R] (Example 12) (11) (3.42 ng/μl), mut-CLuc-F (SEQ ID NO: 8), and SQ-CLuc-F002-rev (SEQ ID NO: 43). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment a(21) in the section 16-3 of Example 16.
The PCR product of the thus obtained fragment a′(238,446) was electrophoresed with 1% agarose, so that it was confirmed to be an approximately 1100-bp DNA fragment. Fragments a′(238,446), a(21) (the section 16-1 of Example 16) and b′′(474,475,476,477,478) (Example 27) were mixed, so that a mixed solution of fragments a(21), a′(238,446) and b′′(474,475,476,477,478) was prepared in a manner similar to that in Example 8.
Overlap PCR was carried out using the above mixed solution of fragments a(21), a′(238,446) and b′′(474,475,476,477,478) as a template. Thus, one long fragment (consisting of the nucleotide sequence ranging from positions 461 to 2663 of SEQ ID NO: 7) was prepared, in which amino acid at target position was substituted. Hereinafter, the DNA fragment is referred to as “fragment c(21,238,446,474,475,476,477,478).”
The composition of the reaction solution for PCR for amplification of fragment c(21,238,446,474,475,476,477,478) was the same as that employed for amplification of fragment c(238,474,475,476,477,478) in Example 26 except for template DNA. The following template DNA was used: the mixed solution (1 μl) of fragments a(21), a′(238,446) and b′′(474,475,476,477,478). Furthermore, the PCR conditions employed herein were the same as those employed for amplification of fragment c(21,249,268) in the section 16-3 of Example 16.
The thus obtained PCR product of fragment c(21,238,446,474,475,476,477,478) was electrophoresed with 0.7% agarose. The experiment was conducted in a manner similar to that in Example 26, so that a mixed solution of fragments c(21,238,446,474,475,476,477,478) and d(238,446,474,475,476,477,478) (Example 26) was prepared.
Next, the emission spectra of the thus obtained clones were measured in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c(21,238,446,474,475,476,477,478) and d(238,446,474,475,476,477,478).
As shown in Table 2, the emission spectral peak of the thus prepared T167K/K375R/Q403P/N404G/T405M/S406L/I407A septuple mutant CLuc (the 1st and the 3rd to the 7th mutant luciferases) was 461 nm.
Example 29
Preparation of Mutant CLuc Via Random Mutagenesis of Q403P/N404G/T405M Triple Mutant CLuc
29-1. Introduction of His-tag into the C-Terminus of Q403P/N404G/T405M Triple Mutant CLuc
A CLuc-(GS)3H6 gene was prepared by ligating an His-tag gene downstream of a CLuc gene regarding triple (Q403P, N404G, and T405M) mutant CLuc (the 4th and the 5th mutant luciferases) with respect to the amino acid sequence shown in SEQ ID NO: 2. Furthermore, amino acid at position 21 within the signal sequence of αCLuc shown in SEQ ID NO: 6 was substituted with leucine.
A DNA fragment consisting of the nucleotide sequence ranging from positions 900 to 2552 of SEQ ID NO: 7 and containing amino acid mutations at positions 403, 404, and 405 is hereinafter referred to as “fragment c′(474,475,476).”
The following oligo DNA primers were used for PCR for amplification of fragment c′(474,475,476): mut-CLuc-F (SEQ ID NO: 8) and c-trm-r: ctagggtgtctccatgctttatgta (SEQ ID NO: 53). Furthermore, the composition of the reaction solution for PCR for amplification of fragment c′(474,475,476) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[Q403P,N404G,T405M] (the section 23-2 of Example 23) (1 μl) (1.82 ng/μl). The PCR conditions employed herein were the same as those employed for amplification of fragment c(475X) in Example 8, except for annealing temperature and extension time. Annealing was carried out at 57° C. and extension was carried out for 2 minutes.
Meanwhile, a linear DNA fragment lacking a portion ranging from positions 967 to 2363 of SEQ ID NO: 7 in the sequence of pCLuRA-TDH3[αP21L,-(GS)3H6] (Example 6) was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d(474,475,476).”
The following primers were used for PCR for amplification of fragment d(474,475,476): vec-CLuc-R (SEQ ID NO: 10) and SQ-CLuc-F003: aagctgaacgactctgcaatagtc (SEQ ID NO: 54). The composition of the reaction solution for PCR for amplification of fragment d(474,475,476) was the same as that employed for amplification of fragment d in Example 8 except for template DNA and primers. The following template DNA was used: pCLuRA-TDH3[αP21L,-(GS)3H6] (Example 6) (1 μl) (1.0 ng/μl). The PCR conditions were the same as those employed for amplification of fragment d in Example 8.
The thus obtained PCR products of fragments c′(474,475,476) and d(474,475,476) were electrophoresed with 0.7% agarose, so that they were confirmed to be an approximately 1700-bp DNA fragment and an approximately 7000-bp DNA fragment, respectively. They were mixed and then a mixed solution of fragments c′(474,475,476) and d(474,475,476) was prepared in a manner similar to that in Example 8.
Next, a clone having, with respect to the amino acid sequence shown in SEQ ID NO: 2, amino acid at position 403 mutated from glutamine to proline, amino acid at position 404 mutated from asparagine to glycine, and amino acid at position 405 mutated from threonine to methionine and having, with respect to the amino acid sequence shown in SEQ ID NO: 6, amino acid at position 21 mutated from proline to leucine and a His-tag gene introduced downstream of the mutant CLuc gene was obtained in a manner similar to that in Example 8 using 10 μl of the mixed solution of fragments c′(474,475,476) and d(474,475,476). A pCLuRA-TDH3 plasmid having, with respect to the amino acid sequence shown in SEQ ID NO: 2, amino acid at position 403 mutated from glutamine to proline, amino acid at position 404 mutated from asparagine to glycine, amino acid at position 405 mutated from threonine to methionine and having, with respect to the amino acid sequence shown in SEQ ID NO: 6, amino acid at position 21 mutated from proline to leucine and a His-tag gene introduced downstream of the mutant CLuc gene, is defined as “pCLuRA-TDH3[αP21L,Q403P,N404G,T405M,-(GS) 3H6].”
29-2. Random Mutagenesis of pCLuRA-TDH3[αP21L,Q403P,N404G,T405M,-(GS)3H6]
pCLuRA-TDH3[αP21L,Q403P,N404G,T405M,-(GS)3H6] was subjected to random mutation. In addition, an explanation is given using the nucleotide numbers (positions) of SEQ ID NO: 23. Mutagenesis was carried out as follows. CLuc was divided into an N-terminal side portion ranging from positions 900 to 1813 and a C-terminal side portion ranging from 1554 to 2699 of SEQ ID NO: 23 and the portions were separately amplified by PCR using different nucleotide concentrations.
The nucleotide sequence ranging from positions 900 to 1813 of SEQ ID NO: 23 was amplified by error prone PCR. The DNA fragment is referred to as “fragment c(474,475,476)-N.” Furthermore, the nucleotide sequence ranging from positions 1554 to 2699 and containing amino acid mutations at positions 403, 404, and 405 of the amino acid sequence shown in SEQ ID NO: 2 was amplified by error prone PCR. The DNA fragment is referred to as “fragment c(474,475,476)-C.”
The composition of the reaction solution for error prone PCR for amplification of fragment c(474,475,476)-N is as follows: Taq DNA polymerase (Roche): 1 μl (5 U/μl); pCLuRA-TDH3[αP21L,-(GS)3H6] (Example 6) plasmid solution (150 ng/μl): 1 μl; 10×PCR buffer w/o Mg2+; for Taq (Roche): 10 μl; 10×dNTP mixture for error prone PCR: 10 μl; 25 mM magnesium chloride: 28 μl; 5 mM manganese chloride: 2.5 μl; mut-CLuc-F (SEQ ID NO: 8): 3 μl; mut-CLuc-NR2 (SEQ ID NO: 9): 3 μl; and sterile water: 41.5 μl. Furthermore, the composition of 10×dNTP mixture for error prone PCR is as follows: 100 mM dCTP: 100 μl; 100 mM dTTP: 100 μl; 100 mM dGTP: 100 μl; 100 mM dATP: 100 μl; and sterile water: 760 μl. PCR was carried out for 30 cycles each consisting of 94° C. for 1 minute (denaturation), 45° C. for 1 minute (annealing), and 72° C. for 1 minute (extension).
Meanwhile, the composition of the reaction solution for error prone PCR for amplification of fragment c(474,475,476)-C was the same as that employed for error prone PCR for amplification of fragment c(474,475,476)-N, except for template DNA, primers, and the amount of sterile water. The following template DNA, oligo DNA primers, and amount of sterile water were used: pCLuRA-TDH3[αP21L,Q403P,N404G,T405M,-(GS)3H6] (the section 29-1 above) plasmid solution (0.5 μl) (288 ng/μl), mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13), and sterile water (42 μl). The PCR conditions were the same as those for error prone PCR for amplification of fragment c(474,475,476)-N.
The thus obtained PCR products of fragments c(474,475,476)-N and c(474,475,476)-C were electrophoresed with 1% agarose, so that an approximately 900-bp c(474,475,476)-N fragment and an approximately 1100-bp c(474,475,476)-C fragment could be confirmed. They were separately subjected to purification using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), phenol extraction, and then ethanol precipitation. The resultants were each dissolved in 10 μl of sterile water (“a solution of fragment c(474,475,476)-N” and “a solution of fragment c(474,475,476)-C,” respectively).
Next, fragments c(474,475,476)-N and c(474,475,476)-C were each amplified by PCR. The thus 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 reaction solution for PCR for amplification of fragment c(474,475,476)-N(2) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA and primers. The following template DNA and oligo DNA primers were used: the solution (1 μl) of fragment c(474,475,476)-N, mut-CLuc-F (SEQ ID NO: 8), and mut-CLuc-NR2 (SEQ ID NO: 9).
Meanwhile, the composition of the reaction solution for PCR for amplification of fragment c(474,475,476)-C(2) was the same as that employed for amplification of fragment c(474,475,476)-N(2), except for template DNA and primers. The following template DNA and oligo DNA primers were used: the solution (1 μl) of fragment c(474,475,476)-C, mut-CLuc-CF1 (SEQ ID NO: 12), and mut-CLuc-R (SEQ ID NO: 13).
Furthermore, the PCR conditions for amplification of fragments c(474,475,476)-N(2) and c(474,475,476)-C(2) were the same as those employed for amplification of fragment c(475X) in Example 8, except for annealing temperature and extension time. Annealing was carried out at 60° C. and extension was carried out for 1 minute and 30 seconds.
The thus obtained PCR products of fragments c(474,475,476)-N(2) and c(474,475,476)-C(2) were electrophoresed with 1% agarose, so that an approximately 900-bp fragment c(474,475,476)-N(2) and an approximately 1100-bp fragment c(474,475,476)-C(2) could be confirmed. They were each subjected to purification using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), phenol extraction, and then ethanol precipitation. The resultants were each dissolved in 20 μl of sterile water (“a solution of fragment c(474,475,476)-N(2)” and “a solution of fragment c(474,475,476)-C(2),” respectively).
Meanwhile, a linear DNA lacking a portion ranging from positions 967 to 1703 of SEQ ID NO: 23 in the sequence of pCLuRA-TDH3[αP21L,-(GS)3H6] (Example 6) was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d(474,475,476)-N.” Furthermore, a linear DNA lacking a portion ranging from 1664 to 2611 of SEQ ID NO: 23 was amplified by PCR. The DNA fragment is referred to as “fragment d(474,475,476)-C.”
The composition of the reaction solution for PCR for amplification of fragment d(474,475,476)-N was the same as that employed for amplification of fragment d in Example 8, except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L,Q403P,N404G,T405M,-(GS) 3H6] (the section 29-1 above) (1 μl) (288 ng/μl), vec-CLuc-R (SEQ ID NO: 10), and SQ-CLuc-NF2 (SEQ ID NO: 11).
Furthermore, the composition of the reaction solution for PCR for amplification of fragment d(474,475,476)-C was the same as that employed for amplification of fragment d(474,475,476)-N, except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L,-(GS)3H6] (Example 6) (1 μl) (150 ng/μl), SQ-CLuc-CR1 (SEQ ID NO: 15), and vec-CLuc-F (SEQ ID NO: 14).
The PCR conditions for amplification of fragments d(474,475,476)-N and d(474,475,476)-C were the same as those employed for amplification of fragment d in Example 8.
The thus obtained PCR products of fragments d(474,475,476)-N and d(474,475,476)-C were electrophoresed with 0.7% agarose, so that an approximately 7000-bp d(474,475,476)-N fragment and an approximately 7000-bp d(474,475,476)-C fragment could be confirmed. They were each subjected to purification using GeneElute MINUS EtBr SPIN COLUMNS (Sigma), phenol extraction, and then ethanol precipitation. The resultants were each dissolved in 10 μl of sterile water (“a solution of fragment d(474,475,476)-N” and “a solution of fragment d(474,475,476)-C,” respectively).
Next, a mixed solution of fragments c(474,475,476)-N(2) and d(474,475,476)-N was prepared by mixing 10 μl of the solution of fragment c(474,475,476)-N(2) and 5 μl of the solution of fragment d(474,475,476)-N. Similarly, a mixed solution of fragments c(474,475,476)-C(2) and d(474,475,476)-C was prepared. Transformation was carried out in a manner similar to that in Example 12 using them, thereby constructing “an N-terminal library of αP21L,Q403P,N404G,T405M,-(GS)3H6 mutant” and “a C-terminal library of αP21L,Q403P,N404G,T405M,-(GS)3H6 mutant,” respectively.
Hereinafter, in a manner similar to that in Example 8, clones suspected of having undergone emission spectral shift were selected and then the emission spectra were measured.
As shown in Table 2, the emission spectral peaks of the thus selected Y280D/R372L/Q403P/N404G/T405M quintuple mutant CLuc and I276N/Q403P/N404G/T405M quadruple mutant CLuc (the 4th and the 5th mutant luciferases) were both 462 nm.
The above pCLuRA-TDH3 plasmid having, with respect to amino acid sequence shown in SEQ ID NO: 2, amino acid at position 280 mutated from tyrosine to aspartic acid, amino acid at position 372 mutated from arginine to leucine, amino acid at position 403 mutated from glutamine to proline, amino acid at position 404 mutated from asparagine to glycine, amino acid at position 405 mutated from threonine to methionine and having, with respect to the amino acid sequence shown in SEQ ID NO: 6, amino acid at position 21 mutated from proline to leucine and a His-tag gene introduced downstream of the CLuc gene is defined as “pCLuRA-TDH3[αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS)3H6].”
Example 30
Preparation of Mutant CLuc Via Random Mutagenesis of Y280D/R372L/Q403P/N404G/T405M Quintuple Mutant CLuc
pCLuRA-TDH3[αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS)3H6] was subjected to random mutation. Mutagenesis was carried out in a manner similar to that in the section 29-2 of Example 29.
In this Example, an explanation is given using nucleotide numbers (positions) of SEQ ID NO: 23. The nucleotide sequence ranging from positions 900 to 1717 of SEQ ID NO: 23 was amplified by error prone PCR. The DNA fragment is referred to as “fragment c(351,443,474,475,476)-N.” Furthermore, the nucleotide sequence ranging from positions 1554 to 2699 and containing amino acid mutations at positions 280, 372, 403, 404, and 405 of the amino acid sequence shown in SEQ ID NO: 2 was amplified by error prone PCR. The DNA fragment is referred to as “fragment c(351,443,474,475,476)-C.”
The composition of the reaction solution for error prone PCR for amplification of fragment c(351,443,474,475,476)-N was the same as that employed for error prone PCR for amplification of fragment c(474,475,476)-C in the section 29-2 of Example 29, except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS) 3H6] (the section 29-2 of Example 29) (0.5 μl) (329 ng/μl), mut-CLuc-F (SEQ ID NO: 8), and K340-rev: gtacggctcgagaagaccttt (SEQ ID NO: 55).
Furthermore, the composition of the reaction solution for error prone PCR for amplification of fragment c(351,443,474,475,476)-C was the same as that for error prone PCR for amplification of fragment c(351,443,474,475,476)-N, except for primers. The following oligo DNA primers were used: mut-CLuc-CF1 (SEQ ID NO: 12) and mut-CLuc-R (SEQ ID NO: 13).
The error prone PCR conditions for amplification of fragments c(351,443,474,475,476)-N and c(351,443,474,475,476)-C were the same as those employed for amplifying fragments c(474,475,476)-N and c(474,475,476)-C, separately in the section 29-2 of Example 29.
A solution of fragment c(351,443,474,475,476)-N and a solution of fragment c(351,443,474,475,476)-C were prepared as follows in a manner similar to that in the section 29-2 of Example 29.
Next, fragments c(351,443,474,475,476)-N and c(351,443,474,475,476)-C were separately amplified by PCR. The thus amplified fragments are referred to as “fragment c(351,443,474,475,476)-N(2)” and “fragment c(351,443,474,475,476)-C (2),” respectively.
The composition of the reaction solution for PCR for amplification of fragment c(351,443,474,475,476)-N(2) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA and primers. The following template DNA and oligo DNA primers were used: a solution of fragment c(351,443,474,475,476)-N, mut-CLuc-F (SEQ ID NO: 8), and K340-rev (SEQ ID NO: 55).
Furthermore, the composition of the reaction solution for PCR for amplification of fragment c(351,443,474,475,476)-C(2) was the same as that employed for amplification of fragment c(474,475,476)-C(2) in the section 29-2 of Example 29, except for template DNA. The following template DNA was used: a solution of fragment c(351,443,474,475,476)-C.
The thus obtained PCR products of fragments c(351,443,474,475,476)-N(2) and c(351,443,474,475,476)-C(2) were electrophoresed with 1% agarose and then a solution of fragment c(474,475,476)-N(2) and a solution of fragment c(474,475,476)-C(2) were prepared in a manner similar to that in the section 29-2 in Example 29.
Meanwhile, a linear DNA lacking a portion ranging from positions 967 to 1703 of SEQ ID NO: 23 in the sequence of pCLuRA-TDH3[αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS)3H6] was amplified by PCR. Hereinafter, the DNA fragment is referred to as “fragment d(351,443,474,475,476)-N.”
The composition of the reaction solution for PCR for amplification of fragment d(351,443,474,475,476)-N was the same as that employed for amplification of fragment d in Example 8, except for template DNA and primers. The following template DNA and oligo DNA primers were used: pCLuRA-TDH3[αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS)3H6], vec-CLuc-R (SEQ ID NO: 10), and SQ-CLuc-NF2 (SEQ ID NO: 11). Furthermore, the PCR conditions were the same as those employed for amplification of fragment d in Example 8.
Hereinafter, a solution of fragment d(351,443,474,475,476)-N was prepared in a manner similar to that in the section 29-2 of Example 29.
Next, a mixed solution of fragments c(351,443,474,475,476)-N(2) and d(351,443,474,475,476)-N was prepared by mixing 10 μl of the solution of fragment c(351,443,474,475,476)-N(2) with 5 μl of the solution of fragment d(351,443,474,475,476)-N. Similarly, a mixed solution of fragments c(351,443,474,475,476)-C(2) and d(474,475,476)-C was prepared by mixing 10 μl of the solution of fragment c(351,443,474,475,476)-C(2) with 5 μl of the solution of fragment d(474,475,476)-C (the section 29-2 of Example 29). Transformation was carried out in a manner similar to that in Example 12 using them, thereby constructing “an N-terminal library of αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS)3H6 mutant” and “a C-terminal library of αP21L,Y280D,R372L,Q403P,N404G,T405M,-(GS) 3H6 mutant,” respectively.
Hereafter, clones suspected of having undergone emission spectral shift were selected in a manner similar to that in Example 8 and then the emission spectra were measured.
Mutant CLucs contained in the thus selected clones are as shown below.
(1) V258A/Y280D/R372L/Q403P/N404G/T405M/E479V septuple mutant CLuc (in the mutant CLuc, a linker sequence (GSGSGS) located between CLuc and a histidine tag contained an amino acid substitution, however, this was thought to have no effect on the emission spectral peak)

(2) R87S/Y280D/R372L/Q403P/N404G/T405M sextuple mutant CLuc

(3) K38R/R79S/Y280D/R372L/Q403P/N404G/T405M septuple mutant CLuc

(4) L191Q/Y280D/R372L/Q403P/N404G/T405M sextuple mutant CLuc

(5) V75E/K126E/M223I/Y280D/R372L/Q403P/N404G/T405M eightfold mutant CLuc

(6) K38I/Y280D/R372L/Q403P/N404G/T405M sextuple mutant CLuc

(7) S45G/E170G/Y280D/R372L/Q403P/N404G/T405M septuple mutant CLuc

All of them correspond to the 4th and the 5th mutant luciferases. The emission spectral peak of each mutant CLuc is shown in the following Table 2.








TABLE 2






Maximum emission spectral


Mutant CLuc
wavelength (nm)







N404G
458


N404S
458


T405M
457


S406L
460


I407A
460


T167K/K375R
460


T167K/Q403P
458


T167K/N404G
460


T167K/T405I
460


M178K/L197P
447


K375R/Q403P
460


K375R/N404G
461


K375R/T405I
463


Q403P/N404G
462


Q403P/T405I
459


N404G/T405I
461


Q403P/N404G/T405I
462


Q403P/N404G/T405M
462


Q403P/N404G/T405M/S406L
461


Q403P/N404G/T405M/S406L/I407A
460


T167K/Q403P/N404G/T405M/S406L/I407A
461


K375R/Q403P/N404G/T405M/S406L/I407A
460


T167K/K375R/Q403P/N404G/T405M/S406L/I407A
461


I276N/Q403P/N404G/T405M
462


Y280D/R372L/Q403P/N404G/T405M
462


V258A/Y280D/R372L/Q403P/N404G/T405M/E479V
463


R87S/Y280D/R372L/Q403P/N404G/T405M
464


K38R/R79S/Y280D/R372L/Q403P/N404G/T405M
462


L191Q/Y280D/R372L/Q403P/N404G/T405M
464


V75E/K126E/M223I/Y280D/R372L/Q403P/N404G/T405M
465


K38I/Y280D/R372L/Q403P/N404G/T405M
463


S45G/E170G/Y280D/R372L/Q403P/N404G/T405M
464









Among the above clones, a pCLuRA-TDH3 plasmid having, with respect to the amino acid sequence shown in SEQ ID NO: 2, amino acid at position 191 mutated from leucine to glutamine, amino acid at position 280 mutated from tyrosine to aspartic acid, amino acid at position 372 mutated from arginine to leucine, amino acid at position 403 mutated from glutamine to proline, amino acid at position 404 mutated from asparagine to glycine, amino acid at position 405 mutated from threonine to methionine and having, with respect to the amino acid sequence shown in SEQ ID NO: 6, amino acid at position 21 mutated from proline to leucine and a His-Tag gene introduced downstream of the mutant CLuc gene, is defined as “pCLuRA-TDH3[αP21L,L191 Q,Y280D,R372L,Q403P,N404G,T405M-(GS)3H6].”
Example 31
Preparation of Mutant CLuc Via Random Mutagenesis of L191Q/Y280D/R372L/Q403P/N404G/T405M Sextuple Mutant CLuc
pCLuRA-TDH3[αP21L,L191Q,Y280D,R372L,Q403P,N404G,T405M-(GS) 3H6] was subjected to random mutation. Mutagenesis was carried out in a manner similar to that in the section 29-2 of Example 29.
In this Example, an explanation is given using the nucleotide positions (numbers) of SEQ ID NO: 23. The nucleotide sequence ranging from positions 900 to 1813 of SEQ ID NO: 23 and containing mutations at positions 191 and 280 of the amino acid sequence shown in SEQ ID NO: 2 was amplified by error prone PCR. The DNA fragment is referred to as “fragment c(262,351,443,474,475,476)-N.”
The composition of the reaction solution for error prone PCR for amplification of fragment c(262,351,443,474,475,476)-N was the same as that employed for error prone PCR for amplification of fragment c(474,475,476)-N in the section 29-2 of Example 29, except for template DNA and the amount of sterile water. The following template DNA and amount of sterile water were used: pCLuRA-TDH3[αP21L,L191Q,Y280D,R372L,Q403P,N404G,T405M-(GS)3H6] (Example 30) (0.5 μl) (298 ng/μl) and 42 μl of sterile water. Furthermore, the error prone PCR conditions for amplification of fragment c(262,351,443,474,475,476)-N were the same as those employed for amplification of fragments c(474,475,476)-N and c(474,475,476)-C in the section 29-2 of Example 29.
Hereafter, a solution of fragment c(262,351,443,474,475,476)-N was prepared in a manner similar to that in the section 29-2 of Example 29.
Meanwhile, the nucleotide sequence ranging from positions 900 to 1663 of SEQ ID NO: 23 and containing a mutation at position 191 of the amino acid sequence shown in SEQ ID NO: 2 was amplified by PCR. The thus amplified fragment is referred to as “fragment c(262,351,443,474,475,476)-N(2).”
The composition of the reaction solution for PCR for amplification of fragment c(262,351,443,474,475,476)-N(2) was the same as that employed for amplification of fragment c(475X) in Example 8, except for template DNA and primers. The following template DNA and oligo DNA primers were used: the solution of fragment c(262,351,443,474,475,476)-N, mut-CLuc-F (SEQ ID NO: 8), and SQ-CLuc-CR1 (SEQ ID NO: 15).
The thus obtained PCR product of fragment c(262,351,443,474,475,476)-N(2) was electrophoresed with 1% agarose, so that an approximately 700-bp fragment c(262,351,443,474,475,476)-N(2) could be confirmed. Hereafter, a solution of fragment c(262,351,443,474,475,476)-N(2) was prepared in a manner similar to that in the section 29-2 of Example 29.
Furthermore, linear DNA lacking positions 1664 to 2611 of SEQ ID NO: 23 in the sequence of pCLuRA-TDH3[αP21L,L191Q,Y280D,R372L,Q403P,N404G,T405M-(GS) 3H6] and containing a mutation at position 191 of the amino acid sequence shown in SEQ ID NO: 2 was amplified by PCR. The DNA fragment is referred to as “fragment d(262,351,443,474,475,476)-C.”
The composition of the reaction solution for amplification of fragment d(262,351,443,474,475,476)-C was the same as that employed for amplification of fragment d(474,475,476)-C in the section 29-2 of Example 29, except for template DNA. The following template DNA was used: pCLuRA-TDH3[αP21L,L191Q,Y280D,R372L,Q403P,N404G,T405M-(GS)3H6]. Furthermore, the PCR conditions for amplification of fragment d(262,351,443,474,475,476)-C were the same as those employed for amplification of fragment d in Example 8.
Hereafter, a solution of fragment d(262,351,443,474,475,476)-C was prepared in a manner similar to that in the section 29-2 of Example 29.
Next, a mixed solution of fragments c(262,351,443,474,475,476)-N(2) and d(351,443,474,475,476)-N was prepared by mixing 10 μl of the solution of fragment c(262,351,443,474,475,476)-N(2) with 5 μl of the solution of fragment d(351,443,474,475,476)-N (Example 30). Similarly, a mixed solution of fragments c(351,443,474,475,476)-N(2) and d(262,351,443,474,475,476) was prepared by mixing 10 μl of the solution of fragment c(351,443,474,475,476)-N(2) (Example 30) with 5 μl of the solution of fragment d(262,351,443,474,475,476)-C. Transformation was carried out using them in a manner similar to that in Example 12, thereby constructing “an N-terminal library of αP21L,L262Q,Y351D,R443L,Q474P,N475G,T476M,-(GS)3H6 mutant” and “a C-terminal library of αP21L,L262Q,Y351D,R443L,Q474P,N475G,T476M,-(GS)3H6 mutant.”
Hereafter, clones suspected of having undergone emission spectral shift were selected in a manner similar to that in Example 8 and then the emission spectra were measured.
The emission spectral peak of the thus selected L191Q/Q235R/Y280D/R372L/Q403P/N404G/T405M septuple mutant CLuc (the 8th mutant luciferase) was 466 nm. Furthermore, the emission spectral peak of M178R/L191Q/Y280D/R372L/Q403P/N404G/T405M septuple mutant CLuc (the 9th mutant luciferase) was 435 nm. The difference between the two mutant CLucs in terms of peak wavelength was 31 nm. Thus, two types of mutant luciferase differing in terms of luminescent color were obtained, which can be sufficiently separated with the use of optical filters and program analysis.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A mutant luciferase selected from the following (a) to (d):
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2, but in which the methionine at position 178 is substituted with another amino acid;
(b) a protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the residue corresponding to the methionine at position 178 in SEQ ID NO: 2 is substituted with another amino acid, and wherein said protein has luciferase activity with an emission spectral peak of 449 nm or less;
(c) a protein comprising residues 19-553 of SEQ ID NO: 2, but in which the methionine at position 178 is substituted with another amino acid; and
(d) a protein comprising an amino acid sequence at least 90% identical to residues 19-553 of SEQ ID NO: 2, wherein the residue corresponding to the methionine at position 178 in SEQ ID NO: 2 is substituted with another amino acid, and wherein said protein has luciferase activity with an emission spectral peak of 449 nm or less.

2. The mutant luciferase according to claim 1, wherein said substitution is with lysine.

3. The mutant luciferase according to claim 1, wherein the emission spectral peak ranges from 420 nm to 449 nm.

4. A mutant luciferase selected from the following (a) to (d):
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2, but in which the methionine at position 178, the leucine at position 191, the tyrosine at position 280, the arginine at position 372, the glutamine at position 403, the asparagine at position 404, and the threonine at position 405, are substituted with other amino acids;
(b) a protein comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the residues corresponding to the methionine at position 178, the leucine at position 191, the tyrosine at position 280, the arginine at position 372, the glutamine at position 403, the asparagine at position 404, and the threonine at position 405 of SEQ ID NO: 2, are substituted with other amino acids, and wherein said protein has luciferase activity with an emission spectral peak of 435 nm or less;
(c) a protein comprising residues 19-553 of SEQ ID NO: 2, but in which the methionine at position 178, the leucine at position 191, the tyrosine at position 280, the arginine at position 372, the glutamine at position 403, the asparagine at position 404, and the threonine at position 405, are substituted with other amino acids; and
(d) a protein comprising an amino acid sequence at least 90% identical to residues 19-553 of SEQ ID NO: 2, wherein the residues corresponding to the methionine at position 178, the leucine at position 191, the tyrosine at position 280, the arginine at position 372, the glutamine at position 403, the asparagine at position 404, and the threonine at position 405 of SEQ ID NO: 2, are substituted with other amino acids, and wherein said protein has luciferase activity with an emission spectral peak of 435 nm or less.

5. A fusion protein, wherein a foreign protein or peptide is linked to the mutant luciferase according to any one of claims 1 and 4.

6. A polynucleotide encoding the mutant luciferase according to any one of claims 1 and 4, or encoding the fusion protein according to claim 5.

7. A recombinant vector comprising the polynucleotide according to claim 6.

8. A transformant comprising the recombinant vector according to claim 7.

9. The transformant according to claim 8, wherein said transformant comprises the polynucleotide of claim 6 operatively linked to a first promoter sequence, and further comprises polynucleotide encoding a protein selected from the following (a) to (c) operatively linked to a second promoter, wherein said first and second promoters are different:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(b) a protein comprising residues 19-553 of SEQ ID NO: 2; and
(c) a fusion protein in which a foreign protein or peptide is linked to the protein of (a) or (b).

10. A method for comparing the transcriptional activity of different promoters, comprising the steps of:
(a) causing a culture or a culture supernatant of the transformant according to claim 9 to come into contact with a luciferin or a derivative thereof; and
(b) measuring the luminescence intensity of the emission spectrum based on the activity of each luciferase.