KR102018248B1 - Mutant Gaussia luciferase with enhanced bioluminescence intensity - Google Patents

Abstract

The present invention relates to Gaussian luciferase variants modified to amplify the bioluminescence intensity of Gaussia Princeps derived luciferases using site specific mutations. More specifically, in the amino acid sequence of G Luc, Gaussia princeps Luciferase ( G Luc), one or more of the 60th, 88th, 89th, 90th, and 103th amino acid is replaced with another amino acid. Cya luciferase single or multiple mutants, polynucleotides encoding the variant sequences, recombinant vectors comprising the polynucleotides, transformants comprising the recombinant vectors, and mutant Gauges with enhanced bioluminescence intensity from the transformants A method of making cya luciferase, and a kit comprising said variant Gaussian luciferase.
G Luc ( Gassia luciferase) is the most widely used secretory luciferase for cell-based reporter assays.It is the smallest of the luciferases from various species, and when combined with other proteins By minimizing the impact of the structure, it has the advantage of being used for effective monitoring. However, the wild-type G Luc has a disadvantage that the size of the light emission signal is not enough.
Accordingly, the present invention can prepare a mutant Gaussian luciferase with improved bioluminescent signal by mutating amino acid residues that play an important role in amplifying the bioluminescent signal of Gaussian luciferase. It can be usefully used.

Description

Mutant Gaussia luciferase with enhanced bioluminescence intensity

The present invention relates to Gaussian luciferase variants modified to amplify the bioluminescence intensity of Gaussia Princeps derived luciferases using site specific mutations. More specifically, Gaussian luciferase ( G Luc, Gaussia A single or multiple mutants of Gaussian luciferase prepared by substituting one or more of the 60th, 88th, 89th, 90th, and 103th amino acids in the amino acid sequence of princeps Luciferase) with other amino acids, and encoding the variant sequence. A polynucleotide, a recombinant vector comprising the polynucleotide, a transformant comprising the recombinant vector, a method of producing a mutant Gaussian luciferase having improved bioluminescence intensity from the transformant, and the mutant Gaussian lucifer A kit comprising a lagase.

Bioluminescence assays using luciferases are similar to fluorescence assays or chemiluminescence because of their high sensitivity, broad linearity and very low background signals. Results often exceed the chemiluminescence assay. Luciferases produce light by catalyzing the oxidation of luciferin substrates (luciferin or coelenterazine). To date, luciferase has been cloned and characterized from various species including firefly, Renilla, copepod and bacteria (WW Lorenz et. al. , Proc. Natl . Acad . Sci . USA. , 88: 4438-4442, 1991). The most widely used luciferases for bioassay are firefly luciferase (FLuc), Renilla luciferase ( R Luc) and recently Gaussia luciferase ( R ). G Luc). FLuc plays a catalyst role for the oxidation of luciferin (luciferin) in the presence of ATP, oxygen (oxygen) and Mg ^{+ 2.} On the other hand, R Luc and Luc G is an ATP-independent and a magnesium (Mg ^{+ 2),} uses coelenterazine as a substrate. R Luc is the most widely used and structurally characterized coelenterazine-dependent luciferases (AM Loening et.al. , J. Mol . Biol . , 374: 1017-1028, 2007 ).

However, recently identified from Gaussia Princeps , a marine copepod G Luc, Gaussia princeps Luciferase) is a coelenterazine-dependent bioluminescent protein, the smallest and most bioluminescent of luciferases derived from various species to date, and the most widely used for cell-based repoter assays. It has been found to attract a lot of attention as a secretory luciferase used (BA Tannous et.al. , Mol . Ther . , 11: 435-443, 2005). G Luc includes live imaging, protein-protein interactions, protein dynamics, monitoring tumor progression and high-throughput screening (HTS). The same bioanalysis has various advantages (CA Maguire et.al. , Anal. Chem . , 81: 7102-7106, 2009). Despite its many benefits, little is known about the structure and functional details of G Luc. In addition, luciferases are not related to evolution and do not share sequence similarity. Meanwhile, several reports emphasize protein engineering efforts to enhance and stabilize R Luc variants rather than naturally occurring forms (AM Loening et.al. , Nat. Methods. , 7: 5-6, 2010). Modified R Luc mutants are widely used to develop new strategies for a variety of biomolecular and high-throughput screening based assays (A. Dragulescu-Andrasi et.al. , Proc . Natl . Acad . Sci . USA. , 108: 12060-12065, 2011).

In the case of G Luc, research and development are being conducted to amplify the intensity of light by several times using gene shuffling (MH Degeling et.al. , Anal. Chem . , 85) . : 3006-3012, 2013). Maguire CA et al. Show G Luc variants exhibiting glow-type light emission kinetics in the presence of Triton X-100, suitable for high-throughput screening (HTS). (Ma Maguire et.al. , Anal. Chem. , 81: 7102-7106, 2009). Welsh JP et al . Reported a double mutant variant that exhibits extended luminescence half-life than wild-type G Luc (JP Welsh et.al. , Biochem). .. Biophys Res Commun, 389: .. 563-568, 2009). As described above, a number of variants genetically engineered into G Luc have been known, but mutant Gaussian luciferase which is satisfactory in terms of luminescence intensity and stability is insufficient. Accordingly, there is a need for development of mutant Gaussian luciferase with improved bioluminescence intensity and stability and substrate specificity.

Thus, the inventors of the crustacean We attempted to analyze the detailed role of various highly conserved and matched amino acids in the luciferase (copepod luciferase). Blast-search was performed to identify homologous crustacean luciferase sequences, and multiple alignments to G Luc were performed to identify highly conserved and matched amino acids. Based on the results of multiple alignments, the role in enhancing bioluminescence intensity was analyzed using conserved amino acids that mutated identical amino acid residues. Single mutants with improved bioluminescent intensity were further combined to construct multiple mutants with maximum bioluminescent intensity.

Against this background, the present inventors have made diligent efforts to develop mutant Gaussian luciferases with amplified bioluminescence intensity, and as a result, sequence-guided mutagenesis analysis based on the alignment of luciferase sequences of various copepods. -guided mutagenesis analysis) yields key candidate amino acids that can significantly amplify bioluminescence intensity, and Gaussian luciferase single or multiple mutants prepared by substituting the derived amino acids for other amino acids are wild-type. The present invention was completed by confirming that the bioluminescence intensity was significantly increased compared to the wild-type.

W.W. Lorenz et al., Proc. Natl. Acad. Sci. USA., 88: 4438-4442, 1991 A.M. Loening et. Al., J. Mol. Biol., 374: 1017-1028, 2007 B.A. Tannous et al., Mol. Ther., 11: 435-443, 2005 C.A. Maguire et al., Anal. Chem., 81: 7102-7106, 2009 A.M. Loening et. Al., Nat. Methods., 7: 5-6, 2010 A. Dragulescu-Andrasi et.al., Proc. Natl. Acad. Sci. USA., 108: 12060-12065, 2011 M.H. Degeling et al., Anal. Chem., 85: 3006-3012, 2013 J.P. Welsh et. Al., Biochem. Biophys. Res. Commun., 389: 563-568, 2009

It is an object of the present invention to provide Gaussian luciferase variants modified to amplify the bioluminescence intensity of Gaussia Princeps derived luciferases using site specific mutations.

Another object of the present invention is to provide a polynucleotide encoding the variant sequence.

Still another object of the present invention is to provide a recombinant vector comprising the polynucleotide.

Still another object of the present invention is to provide a transformant comprising the recombinant vector.

Another object of the present invention is to provide a method for producing a mutant Gaussian luciferase with improved bioluminescence intensity from the transformant.

Still another object of the present invention is to provide a kit comprising the mutated Gaussian luciferase.

In order to achieve the above object, the present invention is Gaussian cyano luciferase 60th, in the amino acid sequence of (G Luc, Gaussia princeps Luciferase) 88 th, 89 th, other one or more amino acid of the 90th and 103rd amino acids Gaussian luciferase prepared by substitution, single or multiple mutants, polynucleotides encoding the variant sequences, recombinant vectors comprising the polynucleotides, transformants comprising the recombinant vectors, bioluminescence from the transformants Provided are a method of making a modified Gaussian luciferase with enhanced strength, and a kit comprising said modified Gaussian luciferase.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention provides a methionine (M), which is the 60th amino acid, and a lysine (K) which is the 88th amino acid, in the amino acid sequence of Gaussian princeps Luciferase ( G Luc) of SEQ ID NO: 1. It provides a Gaussian luciferase variant, characterized in that one or more amino acids of the 89th amino acid (Phenylalanine (F)) and the 103rd amino acid Serine (Srine, S) is mutated.

As used herein, the term "luciferase" is a luminescent protein, and refers to a luminescent enzyme of a luminescent reaction that exhibits a luciferin-luciferase reaction (LL reaction) in bioluminescence. Luciferase is typical of firefly luciferase (Fluc) from Photinus Pyralis, a North American bond, but recently Gaussia luciferase ( G Luc) is the smallest. Bioluminescence is strong and attracting attention.

In the present invention the term " G Lucia Gaussia princeps Luciferase "was identified from Gaussia Princeps Luciferase, the smallest of the luciferases derived from various species to date, has strong bioluminescence, and is the most widely used secretory luciferase for cell-based repoter assays. Known as Aze

Wild-type Gaussian luciferase ( G Luc) is composed of 185 amino acids (gen acid), GenBank accession number is AAG54095.1, represented by SEQ ID NO: 1 in the present invention. Meanwhile, the nucleotide sequence of the wild-type Gaussian luciferase ( G Luc) is represented by SEQ ID NO: 14.

" Gaussia luciferase variants" of the present invention utilize Gaussia princes using site specific mutations. Princeps ) is a variant modified to amplify the bioluminescence intensity of luciferase derived from the amino acid sequence of the wild type Gaussian luciferase, i.e., the 60th, 88th, 89th, 90th, and / Or a Gaussian luciferase single or multiple mutant prepared by substituting the 103rd amino acid residue for another amino acid.

Specifically, the " Gaussia luciferase variant" of the present invention refers to the Gaussian luciferase of SEQ ID NO: 1 ( G Luc, Gaussia In the amino acid sequence of princeps Luciferase, the 60th amino acid Methionine (M), the 88th amino acid Lysine (K), the 89th amino acid Phenylalanine (F) and the 103rd amino acid Serine (S) At least one of the amino acids may be mutated. In addition, the "Gaussia luciferase variant" of the present invention, in addition to the amino acid at the four positions, the Gaussian luciferase of SEQ ID NO: 1 ( G Luc, Gaussia In the amino acid sequence of princeps Luciferase), the 90th amino acid isoleucine (Isoleucine, I) may be further modified.

In one embodiment of the present invention, the following Gaussian luciferase single mutants were first prepared having amino acid mutations in a single position:

A variant having an amino acid sequence in which the methionine (M), which is the 60th amino acid, of the amino acid sequence of SEQ ID NO: 1 is substituted with leucine (Lucine, L) (SEQ ID NO: 2);

A variant having an amino acid sequence in which the 88th amino acid Lysine (K) in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine (Q) (SEQ ID NO: 3);

A variant having an amino acid sequence of phenylalanine (F) which is the 89th amino acid of SEQ ID NO: 1 by tyrosine (Y);

A variant having an amino acid sequence of replacing isoleucine (I), which is the 90th amino acid, in the amino acid sequence of SEQ ID NO: 1 with leucine (Lucine, L) (SEQ ID NO: 5); or

A variant having an amino acid sequence in which Serine (S), which is the 103rd amino acid in the amino acid sequence of SEQ ID NO: 1, was replaced with threonine (T) (SEQ ID NO: 6).

As a result of measuring the bioluminescence intensity using the single mutant, it was confirmed that the bioluminescence signal is improved compared to wild type Gaussian luciferase. In particular, M60L and K88Q of the single mutants showed the highest luminous intensity enhancing effect (Fig. 2).

In another embodiment of the present invention, the following Gaussian luciferase multiple mutant was prepared in which one or more amino acid variations in the single position were combined:

In the amino acid sequence of SEQ ID NO: 1, a variant having an amino acid sequence in which the 60th amino acid Methionine (M) was replaced with Leucine (L) and the 88th amino acid Lysine (K) with Glutamine (Q) ( SEQ ID NO: 7);

In the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of replacing the 60th amino acid Methionine (M) with Leucine (L) and the 90th amino acid Isoleucine (I) with Leucine (L) Variant (SEQ ID NO: 8);

In the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of Lysine (K), which is the 88th amino acid, is substituted with glutamine (Q), and Isoleucine (I), which is the 90th amino acid, is substituted by Leucine (L). Variant (SEQ ID NO: 9);

In the amino acid sequence of SEQ ID NO: 1, the 60th amino acid Methionine (M) is leucine (L), the 88th amino acid Lysine (K) is glutamine (Q), the 90th amino acid isoleucine A variant having an amino acid sequence in which (Isoleucine, I) is substituted with leucine (Leucine, L) (SEQ ID NO: 10);

In the amino acid sequence of SEQ ID NO: 1, the 88th amino acid Lysine (K) is converted into glutamine (Q), the 90th amino acid isoleucine (I) is converted into leucine (Lucine), and the 103rd amino acid serine A variant having an amino acid sequence in which (Serine, S) was substituted with Threonine (T) (SEQ ID NO: 11);

In the amino acid sequence of SEQ ID NO: 1, methionine (M), the 60th amino acid, is leucine (L), lysine (K), the 88th amino acid, is glutamine (Q), and isoleucine, the 90th amino acid. A variant having an amino acid sequence in which (Isoleucine, I) was substituted with leucine (Leucine, L) and serine (S), which is the 103rd amino acid, with threonine (T) (SEQ ID NO: 12); or

In the amino acid sequence of SEQ ID NO: 1, methionine (M), which is the 60th amino acid, is leucine (L), lysine (K), which is the 88th amino acid, is glutamine (Q), and phenylalanine, which is the 89th amino acid. (Phenylalanine, F) is tyrosine (Y), 90th amino acid isoleucine (I) is leucine (L), and 103th amino acid Serine (S) is threonine (T) Variant having an amino acid sequence substituted with (SEQ ID NO: 13).

As a result of measuring the bioluminescence intensity using the multiple mutants, it was confirmed that the bioluminescence signal was significantly increased as compared with wild type Gausia luciferase. In particular, it was confirmed that the amplification of the luminescent activity of the variant combined with M60L among the multiple mutants (Fig. 3).

The Gaussian luciferase variant of the present invention may have an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 2 to 13, but is not limited thereto, and through one or more substitutions, deletions, inversions, translocations, etc. to these sequences. Variants that achieve the effect of the present invention may also be included in the scope of the present invention.

In some cases, the Gaussian luciferase variants of the present invention may have modifications that increase or decrease their physical and chemical properties such that phosphorylation, sulfation, acrylation, glycosylation, It can be modified by methylation, farnesylation, acetylation, amylation, and the like, and the bioluminescent activity of the Gaussian luciferase variant increased by this modification is substantially reduced. As long as it is maintained as such, functional derivatives may be included in the scope of the present invention.

In another aspect, the present invention provides a polynucleotide encoding the Gaussian luciferase variant sequence.

In the present invention, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are DNA or RNA strands of a predetermined length or more, and are Gaussian luciferase variants. It is a polynucleotide encoding a sequence.

The polynucleotide encoding the Gaussian luciferase variant sequence may be modified by the degeneracy of the codon or in consideration of the codons preferred in the organism to express the Gaussian luciferase variant. Various modifications may be made to the coding region within the scope of not changing the amino acid sequence of the cya luciferase variant. Although not limited thereto, the polynucleotide encoding the Gaussian luciferase variant sequence may be a polynucleotide having any one of the nucleotide sequences of SEQ ID NOs: 15 to 26.

In another aspect, the present invention provides a recombinant vector operably linked to a polynucleotide encoding the Gaussian luciferase variant sequence and a transformant into which the recombinant vector is introduced.

As used herein, the term "recombinant vector" refers to a means for expressing a Gaussian luciferase variant by introducing DNA encoding a Gaussian luciferase variant into a host cell, and includes a plasmid vector, a cosmid vector, a bacteriophage vector and Any conventional vector can be used, including viral vectors and the like, and the vector can be readily prepared by those skilled in the art according to any known method using DNA recombination techniques.

Said "operably linked" refers to that expression control sequences are linked to regulate transcription and translation of the polynucleotide sequence encoding the Gaussian luciferase variant sequence, wherein the polynucleotide sequence is controlled under the control of the expression control sequence (including the promoter) Maintaining an accurate reading frame such that a Gaussian luciferase variant that is expressed and encoded by the polynucleotide sequence is produced.

As used herein, the term "transformation" refers to introducing a gene into a host cell so that the gene can be expressed in the host cell, and the transformed gene can be expressed in the host cell, or inserted into a chromosome or chromosome of the host cell. Anything else located is included without limitation.

The "transformer" provided in the present invention can be produced by introducing and transforming the recombinant vector provided in the present invention into a host. The transformation can be carried out by a variety of methods, as long as it can produce a mutant Gaussian luciferase of the present invention that can enhance the bioluminescence signal, it is not particularly limited.

In addition, the host used in the preparation of the transformant is also not particularly limited as long as it can produce the mutant Gaussian luciferase of the present invention, specifically Escherichia genus, Bacilus genus Bacterial cells such as Pseudomonas , Ralstonia , etc .; Yeast cells such as Saccharomyces cerevisiae ; Fungal cells such as Pichia pastoris , and the like.

The transformant may also be used to produce the variant Gaussian luciferase provided by the present invention. Specifically, the mutant Gaussian luciferase can be obtained from the culture of the transformant.

The method of culturing the transformant may be performed using a method well known in the art. Specifically, the culture may be continuously performed in a batch process or an injection batch or repeated fed batch process, but is not limited thereto. The medium used for the cultivation is adapted to meet the requirements of the specific strain in an appropriate manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like.

In one embodiment of the present invention, a pETGLuc_SEP expression plasmid recombinant vector containing a gene encoding a Gaussian luciferase variant sequence is transformed into an E. coli strain BL21 cell, which is then transformed. By culturing, mutant Gaussian luciferase with enhanced bioluminescence intensity was prepared (Examples 1-3).

In another embodiment, the present invention is a Gaussian lucifer mutated to enhance bioluminescence intensity, comprising culturing the transformant and recovering Gaussian luciferase from its culture or culture supernatant. It provides a method for preparing a laase.

At this time, the step of recovering the mutated Gaussian luciferase to enhance the bioluminescence intensity is cell disruption, extraction, affinity chromatography, ion exchange chromatography, gel of the culture cells or culture supernatant obtained from the culture Known purification methods such as filtration chromatography, hydrophobic chromatography, protein precipitation, dialysis and the like can be carried out alone or as a suitable combination, and the identification of the desired protein recovered is known conventional methods such as SDS-PAGE, Western blot and the like. It can be performed using.

In another aspect, the present invention provides a kit comprising the mutated Gaussian luciferase.

Since the modified Gaussian luciferase of the present invention has improved luminescence compared to wild type Gaussian luciferase, it can be usefully used in conventional assay methods or assay kits in which luciferase has been used. Luciferases catalyze luminescent reactions, and the resulting bioluminescence can be used to monitor biological processes in vitro and in vivo .

In addition, the kit of the present invention may further comprise luciferin.

The luciferin may be, but is not limited to, native coelenterazine or h -coelenterazine which is a 2-deoxy derivative of a natural form.

In the present invention, the term "luciferin" refers to a luminescent substrate of a luminescence reaction that exhibits a luciferin-luciferase reaction (LL reaction) in bioluminescence, and is a type of gadban di luciferin and bandidirushin. , Thiatia luciferin and coelenterazine. Oxygen is oxidized by oxygen molecules in the presence of luminescent enzymes to produce an oxidized product (oxyluciferin) in the excited state, and it generates visible light when it becomes the ground state. In summary, the process of luciferin + O ₂ → do.

Kits of the present invention can be prepared from materials and methods commonly used. Kits of the invention can include, for example, sample tubes, plates, instructions for kit users, solutions, buffers, reagents, samples suitable for standardization or control samples.

As another aspect, the present invention provides a method for measuring a bioluminescence signal comprising contacting the Gaussian luciferase variant and luciferin.

The Gaussian luciferase variant and luciferin are as described above.

"Contact" means that the Gaussian luciferase variant of the present invention and luciferin are present in the same reaction system. For example, the Gaussian luciferase variant of the present invention is placed in a container containing luciferin. Adding, adding luciferin to a container containing the Gaussian luciferase variant of the present invention, and mixing the Gaussian luciferase variant of the present invention with luciferin. The reaction conditions can be carried out under the conditions normally used for the luminescence reaction using Gaussian luciferase or under the same conditions (WO99 / 49019, J. Biol . Chem . , 279, 3212-3217 (2004) and the like).

The bioluminescent signal measuring method may include measuring a light emission amount according to the light emission reaction of the Gaussian luciferase variant of the present invention and luciferin. The luciferin may be, but is not limited to, native coelenterazine or h -coelenterazine which is a 2-deoxy derivative of a natural form.

Specifically, as the reaction solvent, Tris-HCl buffer, buffer such as NaCl, water and the like may be used, and the reaction temperature may be 4 to 40 ° C, preferably 4 to 25 ° C. The pH of the reaction solution may be usually 5 to 10, preferably 6 to 9, more preferably 7 to 8.

In one embodiment of the present invention, 50mM Tris and 50mM NaCl (pH 8.0) are used to mix the Gaussian luciferase variant of the present invention, coelenterazine or h -coelenterazine, and the plate reader Bioluminescence intensity was measured (Examples 1-4).

G Luc ( Gassia luciferase) is the most widely used secretory luciferase for cell-based reporter assays.It is the smallest of the luciferases from various species, and when combined with other proteins By minimizing the impact of the structure, it has the advantage of being used for effective monitoring. However, the wild-type G Luc has a disadvantage that the size of the light emission signal is not enough.

Accordingly, the present invention can prepare a mutant Gaussian luciferase with improved bioluminescent signal by mutating amino acid residues that play an important role in amplifying the bioluminescent signal of Gaussian luciferase. It can be usefully used.

1 is a GLuc ( Gaussia) according to an embodiment of the present invention. Figure shows the sequence alignment of two repeated catalytic domains of princeps Luciferase) and other related copepod luciferases. Here, the arrows indicate the amino acids selected for mutagenesis.
FIG. 2 shows the relative bioluminescence intensity and (B) h of G Luc single mutants measured using (A) native coelenterazine as a substrate according to one embodiment of the invention. It is a graph showing the relative bioluminescence intensity of G Luc single mutants measured using h- coelenterazine as a substrate.
FIG. 3 shows the relative bioluminescence intensity of G Luc multiple mutants measured using (A) native coelenterazine as a substrate according to one embodiment of the invention, and (B) h - a graph of the relative bioluminescence intensity of coelenterazine G Luc multiple mutants were measured by using as a substrate a (h -coelenterazine).
4 is used as a substrate (A) wild-type coelenterazine (native coelenterazine) and (B) h, in accordance with an embodiment of the present invention is a diagram showing the structure of coelenterazine (h -coelenterazine).

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Example  1: Preparation of Experimental Materials and Experimental Methods

Example  1-1: Experimental Materials

PCMV- G Luc ( Gaussia The princeps Luciferase plasmid was purchased from Nanolight technology, USA and used for cloning with specific primers. All primers were purchased from Macrogen (Macrogen, Korea). Wild-type (native type) coelenterazine (coelenterazine), and h of 2- deoxy derivative (2-deoxy derivative) of the native-coelenterazine (h -coelenterazine) are both nano-Light Technologies (Nanolight technology, USA ). All other reagents were purchased from commercial sources and the reagents with the highest purity grade were purchased and used.

Example  1-2: sequence Sequence-guided mutagenesis mutagenesis )

First, relative crustaceans To identify the luciferase sequence, G Luc ( Gaussia princeps Luciferase; GenBank accession number_AAG54095.1; SEQ ID NO: 1) Protein-Blast search was performed using an amino acid sequence. Multiple sequence alignments were performed using luciferase sequences derived from BLAST search, and site-directed mutagenesis and characterization were identified. Highly consensus amino acids were selected. GenBank accession numbers for luciferase sequences are as follows; Metridia asymmetrica 1 (MaLuc_1, BAN91823) , M. asymmetric 2 (MaLuc_2, BAN91824), Metridia curticauda 1 (McLuc_1, BAN91825), M. curticauda 2 (McLuc_2, BAN91826), Metridia pacifica 1 (MpLuc_1, BAG48249), M. pacifica 2 (MpLuc_2, BAG48250), Metridia okhotensis 1 (MoLuc_1, BAM11213), M. okhotensis 2 (MoLuc_2, BAL63033), Metridia longa 1 (MlLuc_1, ABW06650), M. longa 2 (MlLuc_2, AAR17541) _1 Pleuromamma abdominalis , BAL63034), P. abdominalis 2 (PaLuc_2, BAL63035), P. scutullata 1 (PsLuc_1, BAN91827), P. scutullata 2 (PsLuc_2, BAN91828), P. xiphias 1 (PxLuc_1, BAN91832uc_2, P. xiphias 2 (Px xiphias 2) , BAN91829), Lucicutia ovaliformis (LoLuc, BAN91831), Heterorhabdus tanneri 1 (HtLuc_1, BAL63039), H. tanneri 2 (HtLuc_2, BAL63040), Heterostylites major 1 (HmLuc_1, BAL63041) and H. major 2 (HmLuc_2, BAL63042).

Site-directed mutagenesis was performed using the pETGLuc-SEP plasmid carrying the G Luc gene with the SEP-tag at the C-terminal. . pETGLuc-SEP was prepared by cloning the G Luc sequence from a commercial PCMV-GLuc vector. PCR was performed to determine T. Rathnayaka et.al. , Biochim . Biophys . Acta . SEP-tag at the C-terminus to increase solubility according to the method described in, 1814, 1775-1778 (2011), 6 Asp residues (Aspartic acid residues) ) Was added, and the resulting GLuc-SEP was cloned into the pET-28 vector. The sequences of all wild-type and mutant were analyzed using sequencing services of Macrogen, Korea.

Example  1-3: G Luc ( Gaussia princeps Luciferase Expression and Purification of Mutants

Transformation of the pETGLuc_SEP expression plasmid into E. coli strain BL21 cells, and 250 ml LBa medium (Luria-Bertani broth) containing 500 g / ml kanamycin The transformed bacteria were incubated at 37 ° C. until the optical density (OD) value reached 0.7 at 600 nm. 0.5 mM IPTG was added to induce protein expression and further incubated at 25 ° C. for 5 hours. Cells were harvested by centrifugation at 7000 rpm for 10 minutes.

The harvested cell pellet was 12.5 ml lysis buffer (50mM Tris, 300mM NaCl, 1mg / ml lysozyme), 1mM phenylmethylsulfonyl fluoride (PMSF) and 0.5% Triton X-100, pH 8.0) The cells were resuspended and sonicated and crushed. The crushed crude cell extract was centrifuged at 14,000 rpm for 20 minutes and the supernatant was filtered, shaking with 1 ml of Ni-NTA beads for 1 hour at 4 ° C. Incubation was performed. The flow-through was removed and the beads washed three times with wash buffer (50 mM Tris, 300 mM NaCl and 20 mM imidazole, pH 8.0). The bound protein, ie Gaussian luciferase, was eluted with a linear gradient with an imidazole concentration in the wash buffer of 0-0.5M. Fractions containing the expressed protein, namely Gaussian luciferase, were dialysis and concentrated with 50 mM Tris and 50 mM NaCl, pH 8.0. Mutant proteins were also purified in a similar manner.

Example  1-4: G Luc ( Gaussia princeps Luciferase ) Characterization of Mutants

Bioluminescence assay was performed using 100 μl of 50 mM Tris and 50 mM NaCl (pH 8.0) containing purified G Luc protein and coelenterazine substrate. It was. Briefly, wild-type (native type) coelenterazine (coelenterazine), or native-type of the 2-deoxy derivatives (2-deoxy derivative) is h - a coelenterazine (h -coelenterazine) solution (solution) 50㎕ each 50 μl of purified G Luc (final concentration 250 nM) was mixed and bioluminescence intensity was immediately measured at 400-600 nm wavelength using a plate reader. Compare mutant protein intensity with wild-type at 482 nm (maximum intensity wavelength, λ _max ) for bioluminescence intensity analysis, and bioluminescence for further characterization Candidates with increased intensity were selected.

Example  2: G Luc ( Gaussia princeps Luciferase ) Selection and Purification of Mutants

Despite its widespread use, the major structural and functional features of G Luc are still unknown. The presence of two repeated catalytic domains, activated when expressed individually, was identified in G Luc (S. Inouye. Et.al. , Biochem . Biophys . Res. Commun . , 365: 96- 101, 2008). By using hydrophobicity search to compare the sequence similarity of the chomophore region of GFP (green fluorescent protein) and coelenterazine, the active site of G Luc ) Is located between 71-140 amino acid, a hydrophilic domain. In addition, several mutations (I90L (the 90th Isoleucine replaced with Leucine) and 8990) with enhanced bioluminescence intensity were proposed (SB Kim et . Al . , Anal. Chem . , 83: 8732-8740, 2011). Recently, molecular directed evolution studies on G Luc also improved the mutants (M60I (60) that exhibit glow-type light emission kinetics. It reported the substitution of Methionine as a second Isoleucine)) (CA Maguire et.al., Anal Chem, 81: 7102-7106, 2009; MH Degeling et.al., Anal Chem, 85:.... 3006-3012 , 2013). In these studies, all mutations were confined to the first domain, and it was reported that mutating the corresponding amino acids in the second domain had no additional effect (MH Degeling et.al. , Anal. Chem . , 85) . : 3006-3012, 2013).

Thus, we continued the functional study using G Luc's BLAST-search and closely from 21 copepod families with ~ 37-73% identity. Related luciferases were identified (FIG. 1). G Luc ( Gaussia Multiple sequence alignments of the copepod luciferase associated with princeps Luciferase confirmed that several highly conserved and consensus positions appeared (FIG. 1). In addition, the focus was on the first repeated domains 27-97 and the consensus position was chosen as the position for mutagenesis (indicated by the arrow in FIG. 1 below). The expression of G Luc in bacterial expression systems is known to be inhibited by the formation of five disulfide bonds. Ten cysteine residues present in G Luc are known to form five disulfide bonds and have been found to be the most conserved of luciferases in crustacean (copepods). Mutations of these cysteine residues are known to cause significant loss of G luc activity (AR Goerke et . Al . , Metab. Eng. , 10: 187-200, 2008).

Several attempts have been made to improve the soluble expression and purification of G Luc in bacterial systems (T. Rathnayaka et.al. , Biochim . Biophys . Acta . , 1814: 1775-1778, 2011; AR Goerke et.al., Metab Eng , 10: 187-200, 2008; T. Rathnayaka et.al., Biochim Biophys Acta, 1804:..... 1902-1907, 2010). Among them, Rathnayaka T et al. Reported that the insertion of SEP-tag (SEP-tag; 9 Aspartic acid (Asp) residues) at the C-terminus significantly increased water-soluble expression (T . Rathnayaka et.al., Biochim Biophys Acta, 1814:... 1775-1778, 2011).

Accordingly, the present inventors cloned G Luc having six aspartic acid (Asp) residues at the C-terminus into a PET-28 vector to express and mutate. Used for mutagenesis. Mutagenesis was performed with pET- G Luc_SEP and then confirmed that all sequences were suitable. Inclusion of SEP-tag elicited water-soluble expression of G Luc in E. coli cells. Purification of wild-type and mutant proteins is described by T. Rathnayaka et.al. , Biochim . Biophys . Acta . , 1814, 1775-1778 (2011). Briefly, 250 ml of cell pellets were sonicated and lysed in lysis buffer, and after centrifugation, the supernatant was loaded onto a Ni-NTA column. The column was washed with wash buffer and the bound protein was eluted with an imidazole concentration gradient. The protein containing fractions were dialysis, concentrated to 10% glycerol and subsequently used. The molecular weight of the purified G Luc observed on SDS-PAGE (Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis) gel was ~ 24 kDa, similar to the molecular weight calculated from the sequence. The average yield was 7.0 mg / l. Like the wild type, all mutant proteins were expressed in soluble form and purified to high purity.

Example  3: Characterization of single mutants

All luminescence measurements were performed using the Thermo Scientific ^™ Varioskan ^™ Flash Multimode Reader with 100 μl of reaction buffer (250 mM Tris and 50 mM NaCl, pH 8.0) containing 250 nM luciferase. It was performed by. First, 250 nM of wild type protein and mutant protein were prepared in 100 μl of reaction buffer. Luminescence intensity was measured between 400-600 nm wavelength immediately after addition of 1 μl of native type coelenterazine.

As a result of measuring the luminescence intensity of the mutants selected in Example 2, five mutants M60L (Methionine (M) at the 60th position of G Luc) leucine (Leucine, L)), K88Q (Lysine, K in G Luc at position 88 to Glutamine, Q), F89Y (Phenylalanine (F) at position 89 in G Luc) Tyrosine, Y)), I90L (Isoleucine, I) at position 90 in G Luc with leucine (L)) and S103T (Serine, S) at position 103 in G Luc The luminescence intensity of threonine (substituted with Th) was about 2-7 times amplified than wild-type G Luc (FIG. 2A). In particular, of the five candidate mutants, K88Q and M60L showed the highest luminescence intensity enhancing effect. The substitution of isoleucine (I) (M60I) at the 60th position (M60) of G Luc is a light emission half-life extending from 2.4 minutes (wild type) to 9.1 minutes in the presence of Triton X-100. life) (CA Maguire. et.al. , Anal. Chem . , 81: 7102-7106, 2009). F89W and I90L have also been previously reported for bioluminescence enhancement in G Luc (SB Kim et . Al . , Anal. Chem . , 83: 8732-8740, 2011). For comparison, the intensity of wild type G Luc at 482 nm (λ _max ) was considered 1.

Further, the second embodiment of the selection of mutants (mutants) of the emission intensity h 2- deoxy derivative (2-deoxy derivative) of the wild-type in-year measured by the coelenterazine (h -coelenterazine) substrate As a result, the luminescence intensity of the five mutants M60L, K88Q, F89Y, I90L and S103T was wild-type G as in the case of using native type coelenterazine as a substrate. Amplified intensity over Luc is shown (FIG. 2B). However, wild-type and mutant-type luciferase of both wild-type 2-deoxy derivative (2-deoxy derivative) is h - coelenterazine (h -coelenterazine) than wild-type (native type) coelenterazine (coelenterazine) When used as a substrate showed a relatively much higher emission intensity.

Single mutant
(individual mutant) Substrate Natural Coelenterazine
(native-type coelenterazine) h -coelenterazine
( h -coelenterazine) Luminescence intensity Wild-type 1.00 ± 0.00 1.00 ± 0.00 L52M (Leu52Met) 0.98 ± 0.29 0.80 ± 0.43 P53S (Pro53Ser) 0.43 ± 0.20 0.30 ± 0.13 E59I (Glu59Ile) 1.43 ± 0.22 1.04 ± 0.22 M60L (Met60Leu) 5.90 ± 0.87 9.13 ± 0.12 R65F (Arg65Phe) 0.52 ± 0.10 0.44 ± 0.09 R65Q (Arg65Gln) 0.92 ± 0.20 0.80 ± 0.22 H79K (His79Lys) 0.53 ± 0.11 0.74 ± 0.10 P84A (Pro84Ala) 0.62 ± 0.02 0.50 ± 0.11 K88E (Lys88Glu) 1.33 ± 0.48 1.50 ± 0.32 K88N (Lys88Asn) 1.92 ± 0.63 1.80 ± 0.52 K88Q (Lys88Gln) 6.80 ± 0.62 5.86 ± 0.76 K88R (Lys88Arg) 0.82 ± 0.12 0.90 ± 0.04 F89Y (Phe89Try) 4.33 ± 0.32 3.30 ± 0.10 I90L (Ile90Leu) 3.99 ± 0.28 3.46 ± 0.40 T96D (Thr96Asp) 1.42 ± 0.33 1.23 ± 0.41 T96S (Thr96Ser) 1.09 ± 0.13 0.92 ± 0.21 S103T (Ser103Thr) 1.43 ± 0.38 2.13 ± 0.32

Example  4: multiple mutants mutnat Evaluation of

To further demonstrate the luminescence intensity amplification effect of single mutants of M60L, K88Q, F89Y, I90L and S103T analyzed in Example 3 above, various combinations were constructed and evaluated for the mutations (FIG. 3).

When using native type coelenterazine as a substrate, the triple mutant (M60L-K88Q-I90L) showed the highest luminescence intensity (˜7-fold) of the tested combinations. This showed a slightly higher luminescence intensity value than the single mutant K88Q (FIG. 3A). On the other hand, the G Luc_5 mutation, which includes all five mutations (M60L, K88Q, F89Y, I90L, and S103T), exhibited about 3-fold increased luminescent activity compared to wild-type G Luc (FIG. 3). A). In addition, it was also confirmed that the multiple mutation emission intensity value was slightly reduced than the single mutation emission intensity value, such as the M60L and K88Q single mutant emission intensity values (FIG. 3A). In particular, all multiple mutations (multiple mutation) is a h-deoxy derivative 2- (2-deoxy derivative) of wild-type - significantly greater than when using a coelenterazine (h -coelenterazine) as a substrate, a single mutation (individual mutation) It was confirmed that excellent luminescent activity was improved (B of FIG. 3). Moderate increase in luminescent activity was confirmed from K88Q-I90L and K88Q-I90L-S103T combinations, including M60L (substituting methionine (M) at position 60 of G Luc with leucine, L). , all multiple mutations (multiple mutants) are wild-type (native type) coelenterazine (coelenterazine) than h - coelenterazine (h -coelenterazine) to the emission intensity was remarkably increased when used as a substrate. Through this, it was confirmed that the combination of M60L is a key mutation for important luminescent activity amplification (Fig. 3B). In particular, h - coelenterazine (h -coelenterazine) the case of using as a substrate, G Luc_5 mutations including the all five mutations (M60L, K88Q, F89Y, I90L and S103T) is wild-type (wild-type) Luc G It showed about 29 times higher luminescent activity as compared to (FIG. 3B). To 4, the native coelenterazine (native coelenterazine) and h - the coelenterazine with the only difference that additional -OH group (-OH group) on a point between the native (h -coelenterazine) Considered, the mutated sites are important for substrate specificity and, therefore, could play an important role in bioluminescence intensity amplification. Unlike other luciferase (luciferase), Luc G are known to exhibit a narrow substrate specificity for a coelenterazine derivative (coelenterazine derivative) (narrow substrate specificity ) (S. Inouye et.al., Protein. Expr. Purif. , 88: 150-156, 2013). Native coelenterazine, but not by (native coelenterazine), h - coelenterazine (h -coelenterazine) G Luc_5 (M60L -K88Q-F89Y-I90L-S103T) strong biological amplification of light emission (bioluminescence) by the Means that the substrate specificity of G Luc is high between native coelenterazine and other coelenterazine analogues. In addition, based on a previously reported hydrophobicity search, the hypothesis that G Luc active sites exist between amino acids 71-140 was re-validated through the above results (SB). kim et.al., Anal Chem, 83: .. 8732-8740, 2011).

Multiple mutants
(multiple mutant) Substrate Natural Coelenterazine
(native-type coelenterazine) h -coelenterazine
( h -coelenterazine) Luminescence intensity Wild-type 1.00 ± 0.00 1.00 ± 0.00 M60L-K88Q
(Met60Leu-Lys88Gln) 3.30 ± 0.57 8.61 ± 0.86 M60L-I90L
(Met60Leu-Ile90Leu) 3.25 ± 0.21 14.82 ± 0.26 K88Q-I90L
(Lys88Gln-Ile90Leu) 2.55 ± 0.21 2.80 ± 0.28 M60L-K88Q-I90L
(Met60Leu-Lys88Gln-Ile90Leu) 6.83 ± 1.15 26.00 ± 1.15 M60L-K88Q-S103T
(Met60Leu-Lys88Gln-Ser103Thr) N / A 14.85 ± 0.49 K88Q-I90L-S103T
(Lys88Gln-Ile90Leu-Ser103Thr) 3.00 ± 0.14 4.30 ± 0.42 M60L-K88Q-I90L-S103T
(Met60Leu-Lys88Gln-Ile90Leu-
Ser103Thr) 3.70 ± 1.40 19.27 ± 1.97 Gluc_5
(M60L-K88Q-F89Y-I90L-S103T; Met60Leu-Lys88Gln-Phe89Try-
Ile90Leu-Ser103Thr) 3.00 ± 0.40 29.00 ± 3.29

<110> HANYANG HAK WON CO LTD <120> Mutant Gaussia luciferase with enhanced bioluminescence intensity <130> P17U10C0207 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_wild type <400> 1 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 2 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_M60L <400> 2 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 3 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_K88Q <400> 3 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 4 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_F89Y <400> 4 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Tyr Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 5 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_I90L <400> 5 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 6 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_S103T <400> 6 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Thr Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 7 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_M60L_K88Q <400> 7 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Ile Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 8 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_M60L_I90L <400> 8 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Lys Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 9 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_K88Q_I90L <400> 9 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 10 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_M60L_K88Q_I90L <400> 10 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 11 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_K88Q_I90L_S103T <400> 11 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Thr Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 12 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_M60L_K88Q_I90L_S103T <400> 12 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Phe Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Thr Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 13 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> GLuc_5 <400> 13 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu   1 5 10 15 Ala Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala              20 25 30 Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro          35 40 45 Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Leu Glu Ala Asn Ala      50 55 60 Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile  65 70 75 80 Lys Cys Thr Pro Lys Met Lys Gln Tyr Leu Pro Gly Arg Cys His Thr                  85 90 95 Tyr Glu Gly Asp Lys Glu Thr Ala Gln Gly Gly Ile Gly Glu Ala Ile             100 105 110 Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu         115 120 125 Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys     130 135 140 Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp 145 150 155 160 Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val                 165 170 175 Asp Lys Ile Lys Gly Ala Gly Gly Asp             180 185 <210> 14 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_wilde type <400> 14 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 15 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_M60L <400> 15 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 16 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_K88Q <400> 16 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 17 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_F89Y <400> 17 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagtacatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 18 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_I90L <400> 18 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 19 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_S103T <400> 19 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagaccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 20 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_M60L_K88Q <400> 20 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcatc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 21 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_M60L_I90L <400> 21 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gaagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 22 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_K88Q_I90L <400> 22 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 23 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_M60L_K88Q_I90L <400> 23 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagtccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 24 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_K88Q_I90L_S103T <400> 24 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagatg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagaccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 25 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_M60L_K88Q_I90L_S103T <400> 25 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagttcctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagaccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555 <210> 26 <211> 555 <212> DNA <213> Artificial Sequence <220> <223> GLuc sequence_5 <400> 26 atgggagtca aagttctgtt tgccctgatc tgcatcgctg tggccgaggc caagcccacc 60 gagaacaacg aagacttcaa catcgtggcc gtggccagca acttcgcgac cacggatctc 120 gatgctgacc gcgggaagtt gcccggcaag aagctgccgc tggaggtgct caaagagctg 180 gaagccaatg cccggaaagc tggctgcacc aggggctgtc tgatctgcct gtcccacatc 240 aagtgcacgc ccaagatgaa gcagtacctc ccaggacgct gccacaccta cgaaggcgac 300 aaagagaccg cacagggcgg cataggcgag gcgatcgtcg acattcctga gattcctggg 360 ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 420 acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 480 ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 540 ggggccggtg gtgac 555

Claims (15)

Gaussian luciferase variant, comprising a variant in which the 88th amino acid Lysine (K) in the amino acid sequence of GLuc, Gaussia princeps Luciferase (SEQ ID NO: 1) is substituted with glutamine (Q) .
The mutant of claim 1, wherein the variant is methionine (M), which is the 60th amino acid in the Gaussian luciferase amino acid sequence of SEQ ID NO. ) Is replaced with Tyrosine (Y), 90th amino acid isoleucine (I) is substituted with leucine (Lucine, L), and 103th amino acid Serine (Srine) is replaced with Threonine (T) The Gaussian luciferase variant, further comprising one or more of the substituted variants.
The Gaussian luciferase variant of claim 1, wherein the Gaussian luciferase variant comprises the amino acid sequence of SEQ ID NO: 3. 6.
The method of claim 2, wherein the variant is selected from the amino acid sequence of SEQ ID 1
a) 60th amino acid Methionine (M) is replaced with Leucine (L), 88th amino acid Lysine (Lysine, K) is replaced with glutamine (Q);
b) 88th amino acid Lysine (K) is replaced by glutamine (Q), 90th amino acid isoleucine (I) by leucine (Lucine, L);
c) The 60th amino acid Methionine (M) is Leucine (L), the 88th amino acid Lysine (K) is Glutamine (Q), the 90th amino acid Isoleucine (I) Substitution with this leucine (Leucine, L);
d) 88th amino acid Lysine (K) is glutamine (Q), 90th amino acid Isoleucine (I) is Leucine (L), 103rd amino acid Serine (S) Substitution with this threonine (T);
e) 60th amino acid Methionine (M) is Leucine (L), 88th amino acid Lysine (K) is glutamine (Q), 90th amino acid Isoleucine (I) With leucine (L), serine (S), the 103rd amino acid, is substituted with threonine (T); or
f) The 60th amino acid Methionine (M) is Leucine (L), the 88th amino acid Lysine (K) is Glutamine (Q), the 89th amino acid Phenylalanine (F) This amino acid sequence is tyrosine (Y), the 90th amino acid isoleucine (I) is replaced with leucine (L), and the 103th amino acid serine (S) is replaced with threonine (T). Gaussian luciferase variant consisting of.
delete The Gaussian luciferase variant of claim 4, wherein the Gaussian luciferase variant consists of the amino acid sequence of SEQ ID NO: 7, 9, 10, 11, 12 or 13. 6.
A polynucleotide encoding the Gaussian luciferase variant sequence of claim 1.
According to claim 7, wherein the polynucleotide is a polynucleotide consisting of any one of the nucleotide sequence of SEQ ID NO: 16, 20, 22 to 26.
A recombinant vector in which the polynucleotide of claim 7 is operably linked.
A transformant having a recombinant vector of claim 9 introduced therein.
A method of producing a Gaussian luciferase, wherein the method comprises culturing the transformant of claim 10 and recovering Gaussian luciferase from the culture or the culture supernatant thereof.
A kit comprising the mutated Gaussian luciferase according to claim 11.
The kit of claim 12, wherein luciferin is further included.
The kit of claim 13, wherein the luciferin is coelenterazine or h -coelenterazine.
The method of claim 1, comprising contacting the Gaussian luciferase variant and luciferin.

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