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WO2018169317A1 - Luciférase de gaussia princeps mutante à intensité de bioluminescence amplifiée - Google Patents

Luciférase de gaussia princeps mutante à intensité de bioluminescence amplifiée Download PDF

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WO2018169317A1
WO2018169317A1 PCT/KR2018/003023 KR2018003023W WO2018169317A1 WO 2018169317 A1 WO2018169317 A1 WO 2018169317A1 KR 2018003023 W KR2018003023 W KR 2018003023W WO 2018169317 A1 WO2018169317 A1 WO 2018169317A1
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amino acid
luciferase
luc
leucine
variant
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김영필
게디비나야쿠마르
이진오
김은혜
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Industry University Cooperation Foundation IUCF HYU
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

Definitions

  • 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).
  • FLuc firefly luciferase
  • R Luc Renilla luciferase
  • R Luc Gaussia luciferase
  • G Luc G Luc
  • FLuc plays a catalyst role for the oxidation of luciferin (luciferin) in the presence of ATP, oxygen (oxygen) and Mg + 2.
  • 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 of coelenterazine-dependent luciferases (AM Loening et.al. , J. Mol . Biol. , 374: 1017-1028, 2007 ).
  • 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.
  • 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.
  • 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.
  • the present invention is one or more amino acids of the 60th, 88th, 89th, 90th and 103rd amino acids in the amino acid sequence of Gaussian lucips ( G Luc, Gaussia princeps Luciferase) 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
  • Gaussian lucips G Luc, Gaussia princeps Luciferase
  • 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
  • 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.
  • M methionine
  • K lysine
  • G Luc Gaussian princeps Luciferase
  • 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) of Photinus Pyralis, a North American bond, but recently Gaussia luciferase ( G Luc) is the smallest. Bioluminescence is strong and attracting attention.
  • 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 reporter assays.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the following Gaussian luciferase single mutants were first prepared having amino acid mutations in a single position:
  • the following Gaussian luciferase multiple mutant was prepared in which one or more amino acid variations in the single position were combined:
  • 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);
  • 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);
  • 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
  • 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);
  • the 88th amino acid Lysine (K) is converted into glutamine (Q)
  • the 90th amino acid isoleucine (I) is converted into leucine (Lucine)
  • 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);
  • M methionine
  • L leucine
  • K lysine
  • Q glutamine
  • 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
  • Phenylalanine, F is tyrosine (Y)
  • 90th amino acid isoleucine (I) is leucine (L)
  • 103th amino acid Serine (S) is threonine (T) Variant having an amino acid sequence substituted with (SEQ ID NO: 13).
  • G Luc_5 mutants including all five mutations were transfected into various cell lines, and a natural 2-deoxy derivative was obtained.
  • the h - nose case of using the binary Ellen TB (h -coelenterazine) as a substrate a bioluminescence intensity was significantly superior as compared to determine the wild-type Luc G (Fig. 7).
  • 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.
  • 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 also be included in the scope of the present invention.
  • the present invention provides a polynucleotide encoding the Gaussian luciferase variant sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a mutant Gaussian luciferase having enhanced bioluminescence intensity was prepared (Examples 1-3).
  • 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.
  • 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.
  • the present invention provides a kit comprising the mutated Gaussian luciferase.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 50mM Tris and 50mM NaCl 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
  • 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.
  • the wild-type G Luc has a disadvantage that the size of the light emission signal is not enough.
  • 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.
  • FIG. 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.
  • 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).
  • 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).
  • G Luc Gaussia
  • G Luc Gaussia
  • FIG. 6 shows pCMW- G Luc wild-type protein expression vector and pCMW- G Luc_5 (M60L-K88Q-F89Y-I90L-S103T) multiple mutants, according to one embodiment of the invention.
  • Figure schematically shows a protein expression vector.
  • FIG. 7 is a diagram illustrating the bioluminescence intensity of G Luc wild-type and the bioluminescence intensity of G Luc_5 multiple mutants in various cell lines according to one embodiment of the present invention.
  • PCMV- G Luc Gaussia The princeps Luciferase plasmid was purchased from Nanolight technology (USA) and used for cloning using 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-guided mutagenesis
  • 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. okartensis 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).
  • pETGLuc-SEP 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.
  • the harvested cell pellets were 12.5 ml of lysis buffer (50 mM Tris, 300 mM NaCl, 1 mg / ml lysozyme), 1 mM PMSF (phenylmethylsulfonyl fluoride) 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).
  • wash buffer 50 mM Tris, 300 mM NaCl and 20 mM imidazole, pH 8.0.
  • the bound protein ie Gaussian luciferase
  • the bound protein 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.
  • Bioluminescence assay was performed using 100 ⁇ l 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 ⁇ l each 50 ⁇ l 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, ⁇ max ) for bioluminescence intensity analysis, and bioluminescence for further characterization Candidates with increased intensity were selected.
  • G Luc 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.
  • 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.
  • G Luc 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).
  • 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).
  • 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).
  • 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)
  • h -coelenterazine wild-type coelenterazine
  • coelenterazine mutant-type coelenterazine
  • 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).
  • 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).
  • 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).
  • all multiple mutations 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).
  • 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).
  • 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.
  • 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).
  • Intracellular G Luc Gaussia G Luc wild-type protein expression vectors were prepared as vectors and controls for protein expression of princeps Luciferase) mutants (FIG. 6).
  • PCMV- G Luc Gaussia The princeps Luciferase plasmid was purchased from Nanolight technology (USA) and used for cloning using specific primers. All primers were purchased from Macrogen (Macrogen, Korea). Primer sequences are shown in Table 3 below, cloning conditions are shown in Table 4 below, and DNA purification was performed using a Gel Extraction kit (Cosmo genetech, cat # CMG0112) according to the manufacturer's instructions.
  • Primer Name primer order SEQ ID NO: GLucWT_F CCC AAG CTT ATG GGA GTC AAA GTT CTG TTT GCC C (34mer) 27 GLucWT_R GCT CTA GAT TAG TCA CCA CCG GCC CCC TTG (30mer) 28 GLuc5_F CCC AAG CTT ATG GGA GTC AAA GTT CTG TTT GCC C (34mer) 29 GLuc5_R GCT CTA GAT TAG TCA CCA CCG GCC CCC TTG (30mer) 30
  • Reactant Reaction pCMV-GLucWT or pCMV-GLuc5 0.5 ⁇ l GLucWT_F primer or GLuc5_F primer (10 ⁇ M) 0.5 ⁇ l (Final con. 100 nM) GLucWT_R primer or GLuc5_R primer (10 ⁇ M) 0.5 ⁇ l (Final con. 100 nM) 2 ⁇ Gotag (Promega, cat # M7423, Final con. 1 ⁇ ) 25 ⁇ l DW 23.5 ⁇ l 50 ⁇ l total PCR conditions: 95 ° C 2 minutes, 95 ° C 30 seconds, 65 ° C 45 seconds, 72 ° C 1 minute, 72 ° C 5 minutes, 30 cycles
  • the plasmid vector and insert DNA were prepared by digesting with a restriction enzyme. At this time, the reaction was carried out at 37 °C for 2 hours.
  • Vector 9 ⁇ l of pCMV3-untagged vector DNA (Sino Biological, HG10741-UT), 9 ⁇ l of HindIII (Takara, cat # 1060A), 9 ⁇ l of XbaI (Takara, cat # 1093A), 10 ⁇ M (Takara, Lot # A4001A, Final con.
  • Example 6 in various cell lines G Luc _5 measurement of bioluminescence intensity of multiple mutants
  • Example 4 h 2- deoxy derivative (2-deoxy derivative) of wild-type - if used as a substrate, a coelenterazine (h -coelenterazine), 5 of all mutations (M60L, K88Q, F89Y, I90L and G Luc_5 mutations, including S103T), showed about 29-fold higher luminescent activity compared to wild-type G Luc.
  • COS7 (origin: kidney, species: monkey green monkey, growth pattern: monolayer, medium: Dulbecco's modified Eagle's Medium) 90%, heat inactivated fetal bovine serum (FBS) 10%), HELA (origin: cervix, uterine, species: human-31 year old black female, growth pattern : Monolayer, medium: DMEM 90%, heat inactivated fetal bovine serum 10%), HT-1080 (species: human-35 year old Caucasian male, growth pattern: monolayer, medium: L-glutamine (300 mg) / l), RPMI1640 90% with 25 mM HEPES and 25 mM NaHCO 3 , 10% heat-inactivated fetal bovine serum, MCF-7 (origin: breast, mammary gland, species: human-69 year old Caucasian) Female, growth pattern: monolayer, medium: L-glutamine (300 mg / L), RPMI1640 90% with 25 mM HEPES and 25 mM H
  • FBS free medium FBS free media; DMEM (Hyclone, cat # 30243.01) or RPMI (Hyclone, cat # SH3002.01)
  • bioluminescence intensity in five cell lines transfected with pCMW- G Luc_5 (M60L-K88Q-F89Y-I90L-S103T) multiple mutants.
  • pCMW- G Luc wild-type As a result of measuring the bioluminescence intensity of each cell line, as shown in FIG. 7, bioluminescence intensity in five cell lines transfected with pCMW- G Luc_5 (M60L-K88Q-F89Y-I90L-S103T) multiple mutants. was found to be relatively higher than the cell line transfected with pCMW- G Luc wild-type.
  • G Luc_5 mutants including all five mutations were transfected into various cell lines, and the native 2-deoxy derivative (2-deoxy) was transfected. derivative) is h - nose case of using the binary Ellen TB (h -coelenterazine) as a substrate, Luc G was confirmed that a bioluminescence intensity significantly superior compared to wild-type. Accordingly, it was found that the G Luc_5 mutant of the present invention can be usefully used for cell-based reporter assay.

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Abstract

La présente invention concerne un mutant de luciférase de Gaussia princeps qui est modifié par mutagenèse spécifique à un site en vue d'amplifier l'intensité de bioluminescence de la luciférase dérivée de Gaussia princeps. Plus particulièrement, la présente invention concerne une luciférase de Gaussia princeps à mutation unique ou multiple, préparée par substitution d'au moins l'un des acides aminés aux positions 60, 88, 89, 90 et 103 de la séquence d'acides aminés de la luciférase de Gaussia princeps (GLuc) par un acide aminé différent, un polynucléotide codant pour la séquence mutante, un vecteur recombinant portant le polynucléotide, un transformant fixant le vecteur recombinant, un procédé de préparation d'une luciférase de Gaussia princeps mutante améliorée en termes d'intensité de bioluminescence à partir du transformant, et un kit comprenant la luciférase de Gaussia princeps mutante. La présente invention peut préparer une luciférase de Gaussia princeps mutante améliorée en termes de signal de bioluminescence par mutation d'un résidu d'acide aminé jouant un rôle critique dans l'amplification du signal de bioluminescence de la luciférase de Gaussia princeps et peut avantageusement appliquer cette dernière à la surveillance de cellules et au criblage de médicaments.
PCT/KR2018/003023 2017-03-15 2018-03-15 Luciférase de gaussia princeps mutante à intensité de bioluminescence amplifiée Ceased WO2018169317A1 (fr)

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WO2024000408A1 (fr) * 2022-06-30 2024-01-04 青岛华大智造普惠科技有限公司 Mutant de luciférase et son utilisation
EP4458958A4 (fr) * 2021-12-31 2025-11-12 Bgi Shenzhen Mutant et ses utilisations

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WO2023109981A2 (fr) * 2023-04-11 2023-06-22 深圳华大智造科技股份有限公司 Nouveau mutant de luciférase de copépode et son application

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4458958A4 (fr) * 2021-12-31 2025-11-12 Bgi Shenzhen Mutant et ses utilisations
WO2024000408A1 (fr) * 2022-06-30 2024-01-04 青岛华大智造普惠科技有限公司 Mutant de luciférase et son utilisation

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