Artificial bioluminescent enzyme
10533231 ยท 2020-01-14
Assignee
Inventors
- Sung Bae Kim (Tsukuba, JP)
- Masaki Torimura (Tsukuba, JP)
- Hiroaki Tao (Tsukuba, JP)
- Tetsuya Nakazato (Tsukuba, JP)
Cpc classification
C12Q1/6897
CHEMISTRY; METALLURGY
C12Y113/12007
CHEMISTRY; METALLURGY
G01N33/535
PHYSICS
G01N33/581
PHYSICS
C12N9/0069
CHEMISTRY; METALLURGY
C07K2319/70
CHEMISTRY; METALLURGY
International classification
C12Q1/6897
CHEMISTRY; METALLURGY
G01N33/535
PHYSICS
Abstract
The invention relates to establishment of a series of artificial luciferases based on artificial amino acid sequences extracted by amino acid alignment of copepod-derived luciferase sequences in a database based on amino acid similarity. The invention provides high luminescence intensity, high luminescence stability, and a spectrum with increased wavelength as luminescence characteristics. A series of artificial luciferases (ALuc) was established. The group of ALucs has superior luminescence characteristics, such as an increase in luminescence intensity, an increase in luminescence stability, or an increase in wavelength of the luminescence spectrum, which were not obtained before. Further, by using the artificial luciferases (ALuc) of the invention, it is possible to provide a novel, superior bioassay system, such as a bioluminescent probe, two-hybrid assay, a luminescent capsule, or the like having improved measurement function.
Claims
1. A nucleic acid encoding a polypeptide having: (a) the amino acid sequence represented by SEQ ID NO: 38, wherein the polypeptide has copepod luciferase activity, and wherein the polypeptide has (i) the amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36, (ii) the amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36, wherein 1-20 amino acids are deleted, substituted, inserted, or added, or (iii) the amino acid sequence having an identity of not less than 90/o with any of amino acid sequences represented by SEQ ID NOs: 11 to 17 and 24 to 36.
2. An expression vector in which the nucleic acid according to claim 1 is inserted in a manner such that the nucleic acid can be expressed.
3. A transformed cell in which the nucleic acid according to claim 1 is introduced in a manner such that the nucleic acid can be expressed.
4. The nucleic acid according to claim 1, wherein the region corresponding to positions 1-71 in the amino acid sequence represented by SEQ ID NO: 38 is represented by SEQ ID NO: 39.
5. The nucleic acid according to claim 1, wherein the region corresponding to positions 1-157 in the amino acid sequence represented by SEQ ID NO: 38 is represented by SEQ ID NO: 40.
6. The nucleic acid according to claim 1, wherein the region corresponding to positions 20-31 in the amino acid sequence represented by SEQ ID NO: 38 includes an antibody recognition site selected from the group consisting of His-tag (HHHHHH) (SEQ ID NO: 5), FLAG-tag (DYKDDDDK) (SEQ ID NO: 6), Myc-tag (EQKLISEEDL) (SEQ ID NO: 7), and HA-tag (YPYDVPDYA) (SEQ ID NO: 8).
Description
BRIEF DESCRIPTION OF DRAWINGS
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(28) (A) A luminescence image of COS-7 cells cultured on a microslide. A significant luminescence image was observed only in living cells expressing ALuc (A16). (B) Luminescence image (left) and a luminescence profile (right) of a COS-7 cell lysate cultured on a microslide.
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(30) Establishment of a series of artificial bioluminescent enzymes containing a functional amino acid sequence (an antigen recognition site (epitope) or an affinity column recognition sequence) and comparison of relative luminescence intensity. (A) Search for an optimal site for fusing each functional amino acid sequence. (B) Relative luminescence intensity of luminescent enzymes established in this research. The luminescence intensity after the secretion was compared using a Promega assay kit.
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(32) The luminescence reaction characteristics of the functional artificial bioluminescent enzymes established in the example (
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(34) (A) An image showing a comparison of luminescence values of different buffer combinations. The image shows that the combination of lysis using a C3 buffer and an assay using an HBSS or TE buffer was most effective.
(35) (B) A graph showing a comparison of luminescence values of different buffer combinations.
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(37) (A) An image showing a comparison of luminescence intensity for different assay buffers.
(38) (B) A graph showing a comparison of luminescence intensity of different assay buffers.
(39) Among assay buffers, PBS buffer, HBSS buffer, and TE-PEG buffer had a good compatibility with C3. The percentages shown above the bars denote the residual luminescence intensity after 8 minutes, relative to the original luminescence intensity (100%).
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(41) (A) Images of luminescence intensity obtained by addition of heavy metals.
(42) (B) A graph showing luminescence intensity obtained by addition of heavy metals.
(43) Aluminum ion, copper ion, iron ion, Mo ion, and zinc ion were found to be effective as additives of assay buffer. The percentages shown above the bars denote the residual luminescence intensity after 8 minutes, relative to the original luminescence intensity (100%)
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(45) (A) The molecular structure and the working mechanism of an integrated-molecule-format probe used in the present example.
(46) (B) Comparison in hormone recognition ability with the use of a buffer containing PEG.
(47) The S/N ratio was higher in the case using a buffer containing 1% PEG than in other cases. The addition of polyethylene glycol mw. 400 (PEG 400) was particularly effective. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(49) (A) The molecular structure and the working mechanism of an integrated-molecule-format probe used in the present example.
(50) (B) A graph showing luminescence intensity obtained by halogen ion addition. A high S/N ratio was obtained by addition of I.sup. ion. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(52) (A) The molecular structure and the working mechanism of an integrated-molecule-format probe used in the present example.
(53) (B) A graph showing luminescence intensity obtained by addition of polysaccharide.
(54) Additions of glucose, sucrose, and glycine were found to be effective. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(56) (A) Molecular structure of cSimgr8 probe. ALuc represents an artificial bioluminescent enzyme (artificial luciferase) that we established. GR LBD represents a stress hormone receptor.
(57) (B) After stimulation of COS-7 cells including pcSimgr8 vector for 20 minutes in the presence or absence of a stress hormone, the luminescence intensity was measured using each one-shot buffer (C14-22). RLU ratio (+/) denotes a luminescence intensity ratio in the presence of stimulation, compared with that in the absence of stimulation. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(59) (A) Molecular structure of cSimgr8 probe ALuc represents an artificial bioluminescent enzyme (artificial luciferase) that we established. GR LBD represents a stress hormone receptor. After stimulating COS-7 cells containing (B) pcSimgr8 vector for 20 minutes in the presence and absence of stress hormone, luminescence intensity was measured using each one-shot buffer (C23-26). RLU ratio (+/) denotes a luminescence intensity ratio in the presence of stimulation, compared with that in the absence of stimulation. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(61) (A) Molecular structure of Leu-rich probe. AR LBD represents a androgen receptor (androgen receptor ligand-binding domain). FLuc represents firefly luciferase.
(62) (B) Cells containing pLeu-rich vector were stimulated with DHT, OHT, E.sub.2, DDT, and PCB for 20 minutes, and compared with the control (0.1% DMSO) stimulation. The results showed that the luminescence response was high with respect to androgen. The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
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(64) (A) Molecular structure of Simer-r2 probe ER LBD represents a estrogen receptor (estrogen receptor ligand-binding domain). CB Red represents a red luminescent enzyme originating from click beetle.
(65) (B) Cells containing pSimer-r2 vector were stimulated with DHT, OHT, E.sub.2, DDT, and PCB for 20 minutes, and compared with the control (0.1% DMSO) stimulation. The results showed that the luminescence response was high with respect to an anti-breast cancer drug (OHT). The symbols and + above the bars denote the presence or absence of androgen (DHT) in the measurement.
[I] ARTIFICIAL BIOLUMINESCENT ENZYME
1. Artificial Luciferases (ALucs) of the Present Invention
(66) (1-1) Copepod Luciferase:
(67) Regarding luminescent marine animals, it is known that marine animals derived from Metridia okhotensis, Pleuromamma abdominalis, Lucicutia ovaliformis, Heterorhabdus tanneri, Heterostylites major, Gaussia princeps, Renilla reniformis, Metridia pacifica, Lucicutia grandis, Lucicutia bicornuta, Pleuromamma xiphias, Pleuromamma scutullata, Haloptilus pseudooxycephalus, Candacia longimana, Candacia columbiae, Candacia bipinnata, Calanus jashnovi, Neocalanus cristatus, Neocalanus flemingeri, Neocalanus plumchrus, Scaphocalanus magnus, Spinocalanus spinipes, Euchaeta marina, Undeuchaeta plumose, Undeuchaeta major, Xanthocalanus kurilensis, Scaphocalanus magnus Gaidius variabilis, Euchirella amoena, Cypridina (Cypridina noctiluca; CLuc), obelin, aqualine, or Oplophorus produce luciferase.
(68) In the present invention, copepod luciferase indicates, among luminescent marine animals, luciferase produced by small crustaceans called copepods that live as luminescent plankton. Specific examples of copepod luciferase include MoLuc1, MoLuc2, PaLuc1, PaLuc2, LoLuc, HtLuc1, HtLuc2, HmLuc1, HmLuc2, Gaussia luciferase (GLuc), Renilla luciferase (RLuc), Metridia luciferase (MLuc, MpLuc1, MpLuc2), Cypridina noctiluca luciferase (CLuc), and the like. Regarding the substrate specificity, copepod luciferase specifically oxidizes coelenterazine. Copepod luciferase generally has an enzymatic property of catalyzing luminescent reaction under a deep-sea environment, i.e., an optimum pH of about 7.5 to 8 and an optimum temperature of about 4 to 10 C.; however, it also catalyzes luminescence under various conditions other than the above. Hereinafter, copepod luciferases refers to luciferases sharing common enzyme activity and structural characteristics with luciferases originating from known copepods. Specifically, copepod luciferases denotes luciferases having an optimum pH of about 5 to 8 and an optimum temperature of about 4 to 25 C., and an enzymatic activity that catalyzes luminescent reaction using coelenterazine as a substrate. The luciferases have two enzymatic activity domains and a secretion signal at their N-terminus, and a molecular weight of about 20 kD (18-28 kD), which is the smallest in the luminescent enzymes. The amino acid sequence homology of copepod luciferases is not less than 50%, and the amino acid sequence structure, such as hydrophilic and hydrophobic patterns, and the position of the enzymatic activity region are similar. Copepod luciferases are luciferases having higher luminescence intensity than other marine organism-derived luciferases.
(69) In the present specification, coelenterazine is not limited to native coelenterazine (native CTZ), but includes various derivatives of native coelenterazine. That is, coelenterazine can also be referred to as coelenterazine-type. Specific examples of coelenterazine include native coelenterazine (Native CTZ), coelenterazine ip (CTZ ip), coelenterazine i (CTZ i), coelenterazine hcp (CTZ hcp), coelenterazine 400A (CTZ 400A), coelenterazine fcp (CTZ fcp), coelenterazine cp (CTZ cp), coelenterazine f (CTZ f), coelenterazine h (CTZ h), coelenterazine n (CTZ n), and the like.
(70) (1-2) Artificial Luciferases (ALucs) of the Present Invention
(71) Since the novel artificial luciferases (ALucs) of the present invention have been discovered based on the amino acid sequences of the copepod luciferases, they have the basic enzyme properties of copepod luciferases, such as the substrate specificity and suitable pH described above. The artificial luciferases of the present invention are also novel artificial luciferases having significantly excellent luminescence characteristics such as luminescence intensity, luminescence in a long wavelength, and luminescence stability.
(72) Examples of the typical artificial luciferase (ALuc) of the present invention include ALuc10 (SEQ ID NO: 11), ALuc15 (SEQ ID NO: 12), ALuc16 (SEQ ID NO: 13), ALuc17 (SEQ ID NO: 24), ALuc18 (SEQ ID NO: 14), ALuc19 (SEQ ID NO: 25), ALuc21 (SEQ ID NO: 26), ALuc22 (SEQ ID NO: 15), ALuc23 (SEQ ID NO: 16), Luc24 (SEQ ID NO: 27), ALuc25 (SEQ ID NO: 17), ALuc26 (SEQ ID NO: 28), ALuc27 (SEQ ID NO: 29), ALuc28 (SEQ ID NO: 30), ALuc29 (SEQ ID NO: 31), ALuc30 (SEQ ID NO: 32), ALuc31 (SEQ ID NO: 33), ALuc32 (SEQ ID NO: 34), ALuc33 (SEQ ID NO: 35), and ALuc34 (SEQ ID NO: 36). The artificial luciferase (ALuc) of the present invention can be expressed as a polypeptide having an amino acid sequence of any one of Items (i) to (iii) below and copepod luciferase activity:
(73) (i) an amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36;
(74) (ii) an amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36 in which one or several amino acids are deleted, substituted, inserted, or added,
(75) (herein several means 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acids);
(76) (iii) an amino acid sequence having an identity of not less than 90% with any of amino acid sequences represented by SEQ ID NOs: 11 to 17 and 24 to 36.
(77) For example, an amino acid sequence having an identity of not less than 95%, not less than 96%, not less than 97%, not less than 98%, not less than 99%, and not less than 99.5% is more preferable.
(78) The amino acid sequences of the artificial luciferases (ALucs) of the present invention have common basic frame structures shown in
(79) (iv) the amino acid sequence represented by SEQ ID NO: 37;
(80) (v) an amino acid sequence represented by SEQ ID NO: 37 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-29 and a region corresponding to positions 214-218;
(81) (iv) the amino acid sequence represented by SEQ ID NO: 38;
(82) (v) an amino acid sequence represented by SEQ ID NO: 38 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-31 and a region corresponding to positions 217-221;
(83) (vi) the amino acid sequence represented by SEQ ID NO: 22; or
(84) (vii) an amino acid sequence represented by SEQ ID NO: 22 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-29 and a region corresponding to positions 211-215.
(85) In the amino acid sequence represented by SEQ ID NO: 22, amino acids from positions 1-20 of the N-terminal side are secretion signals (secretion peptide; SP), and a peptide at positions 211-215 of the C-terminal side is a Glycine rich linker peptide (commonly known as a GS linker). Accordingly, part or all of the amino acids in these regions may be deleted. The same applies to positions 1-20 of the N-terminal side and positions 214 to 218 of the C-terminal side in the amino acid sequence represented by SEQ ID NO: 37, and positions 1-20 of the N-terminal side and positions 217-221 of the C-terminal side in the amino acid sequence represented by SEQ ID NO: 38. In copepod luciferases such as Metridia pacifica luciferase 1 (MpLuc1) and Pleuromamma luciferase, the secretion signals correspond to amino acids at positions 1-18 in Metridia pacifica luciferase 1 (MpLuc1), and correspond to amino acids at positions 1-19 in Pleuromamma luciferase. It is known that these amino acids may be deleted.
(86) As proved in Example 1-21, etc., described below, the function of artificial bioluminescent enzyme is not significantly impaired even when position 20 to 29 in the amino acid sequence represented by SEQ ID NO: 22 (corresponding to a region of positions 20-29 in the amino acid sequence represented by SEQ ID No. 37, and a region of positions 20-31 in the amino acid sequence represented by SEQ ID NO: 38) are substituted with a functional amino acid sequence (e.g., antigen recognition site, affinity chromatography recognition site, or official signal). Accordingly, part or all of the amino acids in this region may be deleted.
(87) In the amino acid sequences represented by SEQ ID NOs: 22, 37, and 28, amino acids represented by Xaa are explained in detail below.
(88) Of the amino acids represented by Xaa in SEQ ID NO: 37, amino acids at positions 3, 20-27, 29, 30, 33, 35, 62-64, 67, 74, 75, 83, 84, 87, 88, 127, 137-145, 147, 156, 158, 185, 188, 199, and 203 may be any amino acids. Of these, amino acids at positions 74-75 and 137-140 may be deleted. Preferably, position 3 is E or G; positions 20-27 are PTENKDDI (SEQ ID NO: 41), ATINEEDI (SEQ ID NO: 42), ATINENFE (SEQ ID NO: 43), HHHHHHHH (SEQ ID NO: 44), EKLISEE (SEQ ID NO: 45), MMYPYDVP (SEQ ID NO: 46), or MMDYKDDD (SEQ ID NO: 47); position 29 is I, L, Y, or K; position 30 is V, D, or A; position 33 is E, G, or A; position 35 is K, S, or G; positions 62-64 are ANS or DAN; position 67 is D or G, positions 75-76 are GG or K (deletion of one residue), or may be deleted; positions 83-84 are LE, KA, or KE; positions 87-88 are KE, IE, LE, or KI; position 127 is E, G, or A; positions 137-145 are IGEA (deletion of four residues, SEQ ID NO: 48), IVGA (deletion of four residues, SEQ ID NO: 49), ITEEE (deletion of three residues, SEQ ID NO: 50), or IGGPIVD (SEQ ID NO: 51); position 147 is D or L; position 156 is D, E, N, F, Y, or W; position 158 is E or L; position 185 is K, F, Y, or W; position 188 is D, A, N, F, Y, or W; position 199 is A or K; and position 203 is S, D, N, F, Y, or W.
(89) Amino acids at positions 13, 16, 36, 148, 171, and 215 are hydrophobic amino acids (for example, V, F, A, or L), and it is preferable that position 13 is V or F; position 16 is V or A; position 36 is F or G; position 148 is I or G; position 168 is V or A; and position 215 is A or L.
(90) Amino acids at positions 5, 65, 73, 99, 117, and 211 are hydrophilic amino acids (for example, Q, K, D, R, H, E, or T), and it is preferable that position 5 is Q or K; position 65 is D or R; position 73 is K, H, R, or E; position 99 is T or H; position 117 is K, E, or Q; and position 211 is K or T.
(91) Amino acids at positions 4, 6, 7, 10, 11, 15, 31, 32, 37-39, 61, 66, 72, 76, 81, 136, 157, and 200 are aliphatic amino acids, and it is preferable that positions 4, 6, 7, 10, 11, 15, 32, 61, 76, 81, and 157 are high molecular weight aliphatic amino acids (e.g., I, V, L, or M), and more preferably, position 4 is I or V; position 6 is V or L; and position 7 is L or I; position 10 is L or V; position 11 is I or L; position 15 is L or V; position 32 is I or V; position 61 is L or V; position 76 is L or M; position 81 is L or M; and position 157 is L or M. It is also preferable that positions 31, 34, 37-39, 66, 72, 136, and 200 are low molecular weight aliphatic amino acids (e.g., A, G, T, or L), and more preferably, position 31 is G, L, or A; position 34 is G or I; position 37 is G, A, S, or F; position 38 is T or F; position 39 is T or A; position 66 is A or G; position 72 is G or may be deleted; position 136 is G or A; and position 200 is T or G.
(92) Amino acids at positions 70, 71, 95, and 108 are positively-charged amino acids (basic amino acids such as K, R or H), and it is preferable that positions 70 and 71 are each R, or may be deleted; position 95 is K or R; and position 108 is H or K.
(93) Amino acids at positions 60 and 208 are negatively-charged amino acids (acidic amino acids such as N, D, Q, or E), and it is preferable that position 60 is N or D, and position 208 is Q or E.
(94) Of the amino acids represented by Xaa in SEQ ID No. 38, amino acids at positions 3, 20-29, 31, 32, 35, 37, 64-66, 69, 76-77, 85-86, 89-90, 129, 140-144, 148-151, 159, 161, 188, 191, 202, and 206 may be any amino acids. Of these, amino acids at positions 22-23, 39-40, 76-77, 140, and 148-151 may be deleted. Preferably, position 3 is E or G; positions 20-29 are PTENKDDI (deletion of two residues, SEQ ID NO: 52), ATINEEDI (deletion of two residues, SEQ ID NO: 53), ATINENFEDI (SEQ ID NO: 54), HHHHHHHH (deletion of two residues, SEQ ID NO: 55), EKLISEE (deletion of two residues, SEQ ID NO: 56), MMYPYDVP (deletion of two residues, SEQ ID NO: 57), or MMDYKDDD (deletion of two residues, SEQ ID NO: 58); position 31 is I, L, Y, or K; position 32 is V or A; position 35 is E or G; position 37 is K or S; positions 64-66 are ANS or DAN; position 69 is D or G; positions 76-77 are GG or K (deletion of one residue), or may be deleted; positions 85-86 are LE, KA, or KE; positions 89-90 are KE, IE, LE, or RI; position 129 is E, G, or A; positions 140-144 are TEEET (SEQ ID NO: 59), GEAI (deletion of one residue, SEQ ID No. 60), or VGAI (deletion of one residue, SEQ ID NO: 61); positions 148-151 are GVLG (SEQ ID NO: 62), GEAI (deletion of one residue, SEQ ID NO: 60), or VGAI (deletion of one residue, SEQ ID NO: 61); positions 148-151 are GVLG (SEQ ID NO: 62) or I (deletion of three residues), or all may be deleted; position 159 is D, E, N, F, Y, or W; position 161 is E or L; position 188 is K, F, Y, or W; position 191 is D, A, N, F, Y, or W; position 202 is A or K; and position 206 is S, D, N, F, Y, or W.
(95) Amino acids at positions 13, 16, 174, and 218 are hydrophobic amino acids (e.g., V, F, A, or L), and it is preferable that position 13 is V or F; position 16 is V or A; position 174 is V or A; and position 218 is A or L.
(96) Amino acids at positions 5, 67, 75, 101, 119, and 214 are hydrophilic amino acids (e.g., Q, K, D, R, H, E, or T), and it is preferable that position 5 is Q or K; position 67 is D or R; position 75 is K, H, R, or E; position 101 is T or H; position 119 is K, E, or Q; and position 211 is K or T.
(97) Amino acids at positions 4, 6, 7, 10, 11, 15, 33, 34, 39-41, 63, 68, 77, 78, 83, 138, 160, and 203 are aliphatic amino acids, and amino acids at positions 39, 40, and 70 may be deleted. It is preferable that positions 4, 6, 7, 10, 11, 15, 34, 63, 78, 83, and 160 are high molecular weight aliphatic amino acids (e.g., I, V, L, or M); however, they may be less-frequently occurring low molecular weight aliphatic amino acids. More preferably, position 4 is I or V; position 6 is V or L; position 7 is L or I; position 10 is L or V; position 11 is I or L; position 15 is L or V; position 34 is I or V; position 63 is L or V; position 78 is L or M; position 83 is L or M; and position 160 is L or M. It is also preferable that positions 33, 39-41, 68, 74, 137, and 203 are low molecular weight aliphatic amino acids (e.g., A, G, or T); however, they may be less-frequently occurring high molecular weight aliphatic amino acids. More preferably, position 33 is G, L, or A; position 39 is G, A, S, or F, or may be deleted; position 40 is T or may be deleted; position 41 is T or A; position 68 is A or G; position 74 is G or may be deleted; position 137 is G or A; and position 203 is T or G.
(98) Amino acids at positions 72, 73, 97, and 110 are positively-charged amino acids (basic amino acids such as K, R or H), and amino acids at positions 72 and 73 may be deleted. It is preferable that positions 72 and 73 are each R, or may be deleted; position 97 is K or R; and position 110 is H or K.
(99) Amino acids at positions 62 and 211 are negatively-charged amino acids (acidic amino acids such as N, D, Q, or E), and it is preferable that position 62 is N or D, and position 211 is Q or E.
(100) Of the amino acids represented by Xaa in SEQ ID NO: 22, amino acids at positions 3, 22, 26, 27, 30, 33, 35, 37-39, 62, 63, 67, 71-75, 87, 127, 138, 140-142, 155, 185, and 197 may be any amino acids. Of these, part or all of the amino acids at positions 71-75 and 140-142 may be deleted. Of hydrophilic amino acids, it is preferable that positions 3, 22, 27, 33, 127, 140, 141, and 155 are E; positions 26, 30, 62, 67, and 185 are D; positions 35 and 87 are K; position 37 is S; positions 38, 39, 138, 142, and 197 are T; position 63 is N; position 71 is R; and position 73 is D or H. Of hydrophobic amino acids, it is preferable that positions 3, 37, 67, 72, 74, 75, 138, and 197 are G; positions 22, 27, and 141 are I; position 30 is V; positions 33, 39, 62, 63, 127, 140, 155, and 185 are A; position 87 is L; and positions 26 and 38 are F.
(101) Amino acids at positions 4, 6, 7, 10, 11, 13, 15, 16, 20, 31, 34, 36, 61, 66, 81, and 168 are hydrophobic amino acids, and it is preferable that position 4 is I or V; position 6 is V or L; position 7 is I or L; position 10 is V or L; position 11 is I or L; and position 13 is V or F; position 15 is V or L; position 16 is V or A; position 20 is A or P; position 31 is L or G; position 34 is I or G; position 36 is F or G; position 61 is V or L; position 66 is A or G; position 81 is L or M; and position 168 is V or A.
(102) Amino acids at positions 5, 24, 25, 60, 64, 65, 70, 95, 108, 153, 200, and 208 are hydrophobic amino acids, and it is preferable that position 5 is Q or K; position 24 is K or E; position 25 is D or N; position 60 is D or N; position 64 is N or S; position 65 is D or R; position 70 is K or R; position 95 is K or R; position 108 is K or H; position 153 is E or D; position 200 is D or S; and position 208 is K, H, or T.
(103) Typical examples of the amino acid sequence represented by SEQ ID NO: 22 include ALuc10, ALuc15, ALuc16, ALuc18, ALuc22, ALuc23, and ALuc25.
(104) One embodiment of the artificial luciferase of the present invention includes the amino acid sequence represented by SEQ ID NO: 39 as the region corresponding to positions 1-71 in the amino acid sequence represented by SEQ ID NO: 38 (corresponding to the region of positions 1-69 in the amino acid sequence represented by SEQ ID NO: 37, and the region of positions 1-69 in the amino acid sequence represented by SEQ ID NO: 22). Typical examples include ALuc15, ALuc16, ALuc17, ALuc18, and ALuc24.
(105) Another embodiment of the artificial luciferase of the present invention includes the amino acid sequence represented by SEQ ID NO: 40 as the region corresponding to positions 1-157 in the amino acid sequence represented by SEQ ID NO: 38 (corresponding to the region of positions 1-155 in the amino acid sequence represented by SEQ ID NO: 37, and the region of positions 1-152 in the amino acid sequence represented by SEQ ID NO: 22). Typical examples include ALuc22, ALuc25, ALuc26, ALuc27, ALuc28, and ALuc29.
(106) Still another embodiment of the artificial luciferase of the present invention includes an antibody recognition site (epitope sequence) therein. Antibody recognition site or epitope sequence can also be referred to as antigen site. Typical examples include ALuc30, ALuc31, ALuc32, and ALuc34.
(107) Specifically, in the artificial luciferase having an antibody recognition site (epitope sequence) therein, a region corresponding to positions 20-31 in SEQ ID NO: 38 or a region corresponding to positions 20-31 in SEQ ID NO: 37 includes an antibody recognition site (epitope sequence). Preferable examples of the antibody recognition site (epitope sequence) include His-tag (HHHHHH) (SEQ ID NO: 5), FLAG-tag (DYKDDDDK) (SEQ ID NO: 6), Myc-tag (EQKLISEEDL) (SEQ ID NO: 7), and HA-tag (YPYDVPDYA) (SEQ ID NO: 8); however, they are not limited thereto.
(108) In an example of the artificial luciferase having an His-tag therein, amino acids at positions 20-31 in SEQ ID NO: 38 or amino acids at positions 20-31 in SEQ ID NO: 37 are all H (His8 sequence). Typical examples include ALuc30 and Aluc31.
(109) In an example of the artificial luciferase having a c-Myc-tag therein, the sequence of the region corresponding to positions 20-31 in SEQ ID NO: 38 or the sequence of the region corresponding to 20 to 31 in SEQ ID NO: 37 is EQKLISEEDL (Myc-tag sequence, SEQ ID NO: 7). Typical examples include ALuc32.
(110) In an example of the artificial luciferase having an HA-tag therein, amino acids at positions 20-31 in SEQ ID NO: 38 or amino acids at positions 20-31 in SEQ ID NO: 37 are YPYDVPDYA (HA-tag sequence, SEQ ID NO: 8). Typical examples include ALuc33.
(111) In an example of the artificial luciferase having a FLAG-tag therein, amino acids at positions 20-31 in SEQ ID NO: 38 or amino acids at positions 20-31 in SEQ ID NO: 37 are DYKDDDDK (FLAG-tag sequence, SEQ ID NO: 6). Typical examples include ALuc34.
2. Establishment of Novel Artificial Luciferase (ALuc) of the Present Invention
(112) (2-1) Luminescence Characteristics Required for Novel Luciferase (ALuc) of the Present Invention
(113) Luminescence characteristics required for copepod luciferase include high luminescence intensity as well as a red-shifted luminescence spectrum, high luminescence stability, heat resistance, and salt tolerance. Since the shift of the luminescence spectrum to the long wavelength side enhances transmittance of luminescence through tissues such as skin tissues or organs, the red-shifted luminescence spectrum is one of the important luminescence characteristics of a reporter protein, which is used as a luminescence enzyme for bioassays or diagnostic probes. Further, because of strong bioluminescence intensity, an effect of detection in various bioassays, even when a small amount of luminescence molecules is used, is expected. Furthermore, when the temporal luminescence stability is ensured, luminescence signal reliability is enhanced, and a reduction in fading during molecular imaging is anticipated. Still further, because they have heat resistance and salt tolerance, luminescence signals are advantageously reliably ensured, even under various bioassay environments. In the comparison of the artificial luciferases (ALucs) of the present invention, the presence or absence of these characteristics is mainly compared to establish more excellent artificial luciferase (ALuc).
(114) (2-2) Method for Establishing Novel Luciferase:
(115) One of the conventional methods for establishing novel luciferase is a method in which mRNA is directly extracted from body fluids of various copepods, the mRNA is converted to DNA using a reverse transcriptase, and the DNA is inserted to an expression vector (e.g., pcDNA3.1 (+)) for expression, followed by evaluation, thus discovering novel luciferase. Another method is a method in which a mutation is introduced into an already established luciferase to produce and enhance new properties. This method is commonly used as a method for establishing a novel mutation. In this method, a known method, such as site-directed mutagenesis (also called the quick-change method), can be suitably used as a nucleotide mutation method (Non-patent Literature 18).
(116) However, none of the above methods ensures the establishment of high-performance artificial luciferase. For example, various novel luciferases have been established from luminescent animals; however, very few of them have properties sufficient for immediate use in industry, and most of them have been forgotten, without being used in practical applications. In very few cases, properties are modified by mutagenesis; however, mutagenesis generally has a low success rate, and there are very few examples of mutagenesis with good results. Specifically, when one mutation is introduced into a protein having 200 amino acids, the success rate is 1/4000 (200 AA20 AA (kinds of amino acids)); accordingly, as the number of amino acid mutations increase, e.g., two mutations, three mutations, etc., the number of times mutations are introduced will be immeasurable, which is not practical.
(117) The present inventors established novel artificial luciferases (ALucs) focusing on the following points.
(118) (2-3) Strategies for Establishment of Artificial Luciferase of the Present Invention
(119) Point 1:
(120) First, a consensus sequence-driven mutagenesis strategy is suggested as a conventional method to understand protein sequences (Non-patent Literature 13). This is a method for analyzing a sequence, in which it is assumed that frequently occurring amino acids obtained from the alignment of similar amino acid sequences in the known database have the most thermodynamically stable effect. However, the alignment method based on the amino acid similarity has a drawback such that the results may be largely influenced by biased selection, or by the number of similar sequences in the database. While keeping this point in mind, the present researchers made the alignment of similar amino acid sequences represented by Example 1-1.
(121) Point 2:
(122) An approach (single sequence alignment; SSA) is also suggested in which the sequence of luciferase is fragmented into two sequences, and the first sequence and the second sequence are aligned to obtain a hint regarding the luminescence characteristics (Non-patent Literature 3). This approach is based on the premise that a luciferase derived from a copepod luminescent animal has two enzyme active sites. By aligning these two enzyme active sites based on the amino acid similarity, the similarity of the enzyme active sites in the first and second sequences can be easily compared. As described above, since there is a hypothesis that the frequency of amino acids is associated with thermodynamic stability, the present inventors intended to form a thermodynamically stable luciferase sequence by increasing the similarity between the first and second sequences.
(123) Point 3:
(124) Luciferases derived from copepods that have been discovered thus far are known to be similar in the central region and the C-terminal side, but have great variation at the N-terminal side. It is also known that luciferases derived from plankton have about 17 amino acids at the N-terminal side as secretion signals. To complete the entire sequence of ALuc by efficiently determining the sequence at the N-terminal side, which is not known, (1) a method in which similar amino acid sequences in the known database are aligned based on amino acid similarity to extract frequently occurring amino acids is used in combination with (2) a sequence analysis using known software (PSORTII) for looking up the properties of the extracted amino acid sequence to finally determine various candidates for the sequence of ALuc at the N-terminal side.
(125) For example, the properties of an artificially produced sequence at the N-terminal side were examined using PSORTII, and localization shown below is predicted (examples).
(126) N-terminal side of ALuc2
(127) 0%: extracellular
(128) 22.2%: cytosol
(129) 33.3%: ER
(130) N-terminal side of ALuc3
(131) 67%: extracellular
(132) 11.1%: cyto
(133) 11.1%: ER
(134) SP1
(135) 44.4%: endoplasmic reticulum
(136) 33.3%: mitochondrial
(137) 11.1%: Golgi
(138) 11.1%: nuclear
(139) SP2
(140) 33.3%: extracellular, including cell wall
(141) 22.2%: vacuolar
(142) 22.2%: cytoplasmic
(143) 22.2%: endoplasmic reticulum
(144) SP4
(145) 55.6%: extracellular, including cell wall
(146) 22.2%: endoplasmic reticulum
(147) 11.1%: cytoplasmic
(148) 11.1%: vacuolar
(149) Point 4:
(150) The thus-far established length of the amino acid sequence of luciferase derived from copepods varies, and the molecular weight thereof also varies in the range of 20 to 36 kD; such variations are mainly attributable to the varied N-terminal side sequence. In the present invention, to determine the N-terminal side sequence, the N-terminal side amino acid sequence is, under the principle of extraction of frequently occurring amino acids, constructed by making groups, i.e., a group having a relatively short N-terminal side sequence (ALuc5-7), and a group having a relatively long N-terminal side sequence (ALuc2-3 and ALuc8-25); the entire amino acid sequence of the artificial luciferase (ALuc) is thereby determined.
(151) (2-4) Synthesis of Artificial Luciferases (ALucs) of the Present Invention
(152) Amino acid sequence determination is conducted according to the strategies of points 1 to 4 described above, thus producing various novel candidate sequences. For actual expression of these amino acid sequences, a gene codon corresponding to each amino acid is applied based on the gene codon table. For advantageous expression in mammalian cells, specifically, for suitable expression in mouse cells, codons are determined. One example of the nucleotide sequence is shown as SEQ ID NO: 23.
(153) A plurality of restriction enzyme sites are introduced into the gene sequences, and synthesis is requested from a manufacturer (Operon) specializing in gene synthesis. By using synthesis genes encoding artificial luciferase (ALuc), which are obtained after being inserted into vectors, subcloned vectors inserted into pcDNA3.1 (+) produced by Invitrogen are produced. The vectors are introduced into African green monkey kidney-derived COS-7 cells, and the luminescence characteristics of the resulting artificial luciferases (ALucs) are measured using various spectroscopes (e.g., luminometer (GloMax 20/20 n; Promega), spectrophotometer (AB-1850; ATTO), image analyzer (LAS-4000; FujiFilm), and microplate reader (Corona)). Their enzymatic activities are evaluated according to the method described in section (3-1) below, and the results are fed back to the amino acid sequences to thereby establish the novel artificial luciferases (ALucs) of the present invention.
3. Enzymatic Activity of Artificial Luciferase (ALuc) of the Present Invention
(154) (3-1) Enzymatic Activity Confirmation Method
(155) The enzymatic activity of ALuc can be examined, for example, according to the following method.
(156) First, using a known lipid reagent for gene introduction, an expression vector encoding ALuc is introduced into African monkey-derived COS-7 cells; as a control, an expression vector having a known GLuc without any mutation into the cells is also introduced in the same manner. At a predetermined time (from 10 to 20 hours, for example, 16 hours) after the introduction of the vector, a cell lysate is prepared using a known lysis reagent.
(157) Thereafter, the cell lysate is mixed with a known substrate solution containing coelenterazine, and its color intensity, temporal stability in luminescence, etc., are measured.
(158) The luminescence intensity may be found by measuring the intensity at a specific wavelength using a known luminescence spectrophotometer after addition of a known substrate. By performing the measurement every minute, the temporal stability in luminescence can be evaluated. To measure a shift to a longer wavelength, scanning of the entire wavelength is necessary.
(159) (3-2) Characteristics of Enzymatic Activity of Artificial Luciferase (ALuc) of the Present Invention
(160) Typical examples of the artificial luciferase (ALuc) of the present invention include ALuc10 (SEQ ID NO: 11), ALuc15 (SEQ ID NO: 12), ALuc16 (SEQ ID NO: 13), ALuc17 (SEQ ID NO: 24), ALuc18 (SEQ ID NO: 14), ALuc19 (SEQ ID NO: 25), ALuc21 (SEQ ID NO: 26), ALuc22 (SEQ ID NO: 15), ALuc23 (SEQ ID NO: 16), ALuc24 (SEQ ID NO: 27), ALuc25 (SEQ ID NO: 17), ALuc26 (SEQ ID NO: 28), ALuc27 (SEQ ID NO: 29), ALuc28 (SEQ ID NO: 30), ALuc29 (SEQ ID NO: 31), ALuc30 (SEQ ID NO: 32), ALuc31 (SEQ ID NO: 33), ALuc32 (SEQ ID NO: 34), ALuc33 (SEQ ID NO: 35), and ALuc34 (SEQ ID NO: 36).
(161) Characteristics of enzymatic activity commonly observed in conventional copepod luciferases are as follows.
(162) (1) Exhibiting transient high-intensity light and poor luminescence stability,
(163) (2) Having a secretion signal at the N-terminal side,
(164) (3) The size of the luminescence enzyme being smaller than that of other luminescence enzymes, and
(165) (4) Commonly exhibiting blue light (480 nm).
(166) The ALuc series of the present invention maintain characteristics (2) and (3), but have much higher luminescence stability (Item (1) above) than conventional copepod luciferases. In particular, ALuc15, ALuc16, ALuc17, ALuc18, ALuc19, ALuc20, ALuc21, ALuc22, ALuc23, ALuc24, ALuc25, ALuc26, ALuc27, ALuc28, ALuc29, ALuc30, ALuc31, ALuc32, ALuc33, and ALuc34 exhibit remarkably stable luminescence signals. Regarding the luminescent color (Item (4) above), ALuc15, ALuc16, ALuc17, ALuc18, ALuc19, ALuc20, ALuc21, ALuc22, ALuc23, ALuc24, ALuc25, ALuc26, ALuc27, ALuc28, ALuc29, ALuc30, ALuc31, ALuc32 ALuc33 and ALuc34 all exhibit luminescence spectra shifted to the long wavelength (green or yellow).
(167) In view of the above, the present invention is confirmed to produce artificial luminescent enzymes of great promise that maintain the advantageous features of conventional copepod luciferases while overcoming common problems of conventional copepod luciferases.
4. Functional Improvement of Artificial Luciferase (ALuc) of the Present Invention
(168) The usages of the artificial luciferase (ALuc) of the present invention typically include those as a luminescent enzyme component of a known bioluminescent probe, and, owing to its high luminance and stable luminescence signal, as a substitute for a reporter gene for fluorescent imaging in vivo. The present invention is mainly used in mammals such as humans in vivo, or in mammalian cells in vitro.
(169) Accordingly, the advantageous modifications for improving other functions include modification of the codons corresponding to the amino acid into codons suitable for host organisms for easy expression, and an improvement of expression promoters for indirect functional improvement. Further, by linking a functional peptide to an N- or C-terminus of artificial luciferase (ALuc) of the present invention, various additional functions can be expected. For example, by linking a membrane localization signal (MLS) to the N- or C-terminus, the ALuc can be localized in the plasma membrane. In this case, the secretion signal at the N-terminal side (positions 1-20, or part of the sequence) derived from ALuc may be present or absent; however, since the secretion signal is transferred across endoplasmic reticulum, the folding efficiency of an ALuc-containing fusion protein can sometimes be increased. In the present invention, when two or more types of peptides, including a signal peptide, are linked, the length, reading frame, etc., are adjusted using a well-known suitable linker, even when the linker is not specified. Localization of ALuc in the plasma membrane allows smooth external supply of the substrate or oxygen. Thus, a luminescent probe (e.g., luminescent capsule) containing ALuc as a base can quickly respond to the external signal (see Example 1-8). The present invention adopts the above as required. The modification strategies for improving functions are specifically described below; however, the present invention is not limited to these examples.
5. Application of Luciferase (ALuc) of the Present Invention to Reporter Analysis Method
(170) (5-1) Reporter Analysis Method of the Present Invention
(171) The ALuc of the present invention and the gene thereof can be preferably used as a reporter protein or a reporter gene in reporter analysis methods.
(172) The reporter protein or reporter gene used in the present invention indicate a luminescent label used for examining the behavior of a target protein or a target gene in cells in response to external stimulus. The reporter analysis method in the present invention is an analysis wherein the behavior of a target protein or a target gene in cells in response to external stimulus is observed in view of the luminescence by ALuc, luminescence amount, luminescence timing, or luminescence site, by using the ALuc of the present invention or its gene as a reporter protein or reporter gene. Specifically, the reporter analysis method is a method for qualitatively or quantitatively measuring the expression site, expression timing, or expression amount of the target gene as the luminescence site, luminescence timing, or luminescence amount of reporter protein ALuc.
(173) More specifically, the reporter protein is typically used as a fusion protein by fusing it with the N- or C-terminus of the target protein; however, reporter proteins bisected into the N-terminal side and the C-terminal side are fused with the target protein in a direct manner or via other peptide sequence. The reporter protein is typically used for examining the behavior of the target protein after expression, by linking it to the 5- or 3 terminus of the target gene to form a chimera gene. Similarly, the reporter gene can be bisected, with one part linked to the 5-terminus of the target gene, and the other linked to the 3-terminus of the target gene; or both can be inserted into the target gene for use.
(174) The reporter protein of the present invention can be described as follows using the definition of ALuc above.
(175) The reporter protein comprising a polypeptide having an amino acid sequence represented by any one of following items (i) to (vii) and having copepod luciferase activity;
(176) (i) an amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36;
(177) (ii) an amino acid sequence represented by any of SEQ ID NOs: 11 to 17 and 24 to 36 in which one or several amino acids are deleted, substituted, inserted, or added (herein several means 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acids);
(178) (iii) an amino acid sequence having an identity of not less than 90% with any of amino acid sequences represented by SEQ ID NOs: 11 to 17 and 24 to 36;
(179) (iv) the amino acid sequence represented by SEQ ID NO: 37;
(180) (v) an amino acid sequence represented by SEQ ID NO: 37 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-29 and a region corresponding to positions 214-218;
(181) (vi) the amino acid sequence represented by SEQ ID NO: 38;
(182) (vii) an amino acid sequence represented by SEQ ID NO: 38 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-31 and a region corresponding to positions 217-221;
(183) (viii) the amino acid sequence represented by SEQ ID NO: 22; or
(184) (ix) an amino acid sequence represented by SEQ ID NO: 22 in which one or more amino acids are deleted in at least one of a region corresponding to positions 1-29 and a region corresponding to positions 211-215.
(185) When the reporter protein of the present invention is used in in vivo conditions, e.g., in a living body, the reporter gene comprising a nucleic acid encoding the amino acid sequence represented by the above (i) to (ix) is linked with a target gene, and incorporated into a vector, etc., thus introducing the vector into target cells.
(186) Hereinafter, the reporter analysis method of the present invention is categorized into three groups: basic, inducible, and activatable, which are disclosed in Non-patent Literature 16 of Niu et al.; and application of the ALuc of the present invention to each analysis method is explained. Herein, the basic method is the simplest reporter analysis system in which ALuc is linked with each subject protein for labeling. Typical examples include a bioluminescent enzyme fusion protein that is linked with an antibody (i.e., bioluminescent enzyme label antibody). The inducible method differs from the basic method in that the expression of the reporter is controlled by a promoter. Typical examples include so-called reporter gene assays and two hybrid assays (reporter is expressed depending on stimulus) in addition to a bioluminescence resonance energy transfer (BRET) method. The activatable method is a reporter analysis method utilizing the mechanism wherein the reporter itself actively reacts in response to ligand stimulation to illuminate. Typical examples include an integrated-molecule-format bioluminescent probe and a luminescent capsule. This method can also be applied to a protein complementation assay (PCA), protein splicing assay (PSA), etc.
(187) (5-2) Basic Method
(188) When the ALuc of the present invention is applied to a basic method as a reporter protein, a fusion protein in which the ALuc is simply linked with a target protein may be produced. The basic method differs from the other reporter analysis methods in that expression during the production of the fusion protein is performed by using an uncontrolled-type promoter.
(189) In the present specification, the fusion protein includes (i) a fusion protein integrally expressed from a gene encoding a fusion protein containing a reporter protein, which is ALuc, and a target protein or a peptide recognizing the target protein; and (ii) a fusion protein obtained by separately expressing a reporter protein, which is ALuc, and a target protein or a peptide recognizing the target protein, and linking them by a chemical reaction. Examples of the means for linking separately expressed proteins, etc., by a chemical reaction include linking using a cross linker, linking using an avidin-biotin binding ability, binding using chemical reactivity of amino acid residues, and the like.
(190) A bioluminescent fusion protein that binds to a typical antibody is hereby explained. A bioluminescent fusion protein is completed by producing a chimera DNA in which an ALuc gene is linked with the upstream or downstream of cDNA of antibody single chain variable fragment (scFv), and introducing the DNA into a suitable expression vector. The reporter analysis method of this embodiment is shown in Example 1-15 or Example 1-16 of the present specification.
(191) (5-3) Inducible Method
(192) Application of a bioluminescent enzyme to an inducible method as a reporter protein has been employed for analyzing the expression timing and expression amount of genes obtained upon the production of recombination protein using recombinant DAN technology. In particular, a bioluminescent enzyme has been widely used as an index indicating the expression timing and expression amount change in response to external stimulus. Examples of analysis systems included in inducible methods include reporter gene assays, yeast two-hybrid assays, mammalian two-hybrid assays, protein splicing assays (PSA), protein complementation assays (PCA), circular permutation assays, bioluminescence resonance energy transfer assays (BRET), and the like. Use of the ALuc of the present invention as a reporter gene essential for these analysis systems remarkably improves assay measurement performance. The reporter analysis method of this embodiment is shown in Example 1-9 of the present specification.
(193) Hereinafter, the reporter gene assay and the two-hybrid assay, which are typical inducible method analysis systems, are explained in detail.
(194) (i) Reporter Gene Assay
(195) Although reporter gene assays have been widely used as means for analyzing activation of transcription factors in response to external stimulus and gene expression regulation, they are typically used for detecting endocrine disruptors (environmental hormones) that disturb signal transduction via nuclear receptors. The expression of a target gene (e.g., hormonal response gene) involving signal transduction via nuclear receptors is caused when the complex of a ligand and a receptor binds to a cis region (hormone-response element) that regulates the transcription of the gene. This is an assay in which a plasmid that contains a reporter gene such as luciferase at the downstream of the cis region of each hormone-response gene is introduced into cells, and the amount of the hormone molecule, which is to be a ligand, or the amount of the endocrine disruptor is detected by the amount of bioluminescence.
(196) Examples of host cells used herein include yeast cells, bacteria cells such as Escherichia coli, and insect cells, as well as mammalian cells such as COS cell, CHO-K1 cell, HeLa cell, HEK293 cell, and NIH3T3 cell used for general gene recombination. The present invention is mainly used in mammals, such as humans in vivo, or in mammalian cells in vitro.
(197) In the reporter gene assay, firefly luciferase that has been widely used has the following drawbacks: (i) due to its large molecular weight, the start of expression takes a long period of time, thereby imposing a great burden on the host cells, and (ii) due to the low luminescence intensity of firefly luciferase, it generally takes 1 to 2 days after stimulation to obtain a sufficient accumulation of luciferase (reporter). However, by selecting the ALuc of the present invention as a reporter protein, these problems are overcome.
(198) Since the use of the ALuc of the present invention as a reporter protein ensures a significantly high luminescence intensity of the reporter, it has an advantage of very prompt measurement after the stimulation. Accordingly, the measurement time can be greatly reduced compared to conventional reporter proteins while ensuring high temporal stability in luminescence, thereby enabling luminescence measurement even for a cell strain with insufficient gene introduction. Further, since the red-shifted luminescence improves transmittance through the plasma membrane or skin, the background level is reduced, and high measurement accuracy can be attained.
(199) More specifically, the ALuc of the present invention is employed in these reporter gene assays in such a manner that the luminescent enzyme is linked to a known eukaryotic cell expression vector containing a special promoter in an upstream portion, and the vector is then introduced into a eukaryotic cell. After a predetermined time, the measurement is performed either in the presence or absence of signal (stimulation) (Non-patent Literature 20). The known pTransLucent vector can be used as this expression vector for reporter gene assay that can carry the ALuc of the present invention; the ALuc can easily be incorporated therein using a known method.
(200) (ii) Two-Hybrid Method
(201) The two-hybrid method is one of the techniques for discovering protein-protein interactions. In 1989, a yeast two-hybrid (Y2H) system using a Saccharomyces cerevisiae yeast was first established. This method utilizes the fact that the DNA binding domain (GAL4 DBD) and the transcriptional activation domain (TA) of GAL4 protein, which is a transcriptional activator, are separable. Fused GAL4 DBD and protein A (bait) are expressed as a fusion protein, and simultaneously, fused transcriptional activation domain (TA) and protein B (prey) are expressed in the cell as a fusion protein. Thus, interaction between proteins A and B can be observed. When proteins A and B bind, DBD approaches TA and binds to the UASG base sequence, which promotes the expression of the reporter gene that is linked to the downstream of the sequence. If the reporter gene is luciferase, the compatibility of proteins A and B can be detected by monitoring bioluminescence in the presence of its specific substrate. This enables screening of protein and peptide that interact with protein A (bait). The protein B (prey) used herein can be supplied from an expression library.
(202) Examples of host cells include, in addition to yeast cells, bacteria such as Escherichia coli, mammalian cells, and insect cells. Other than GAL4 DBD, which is a transcriptional activator derived from a yeast, LexA etc., which is a repressor protein derived from Escherichia coli, can be used. A DNA encoding such a protein is linked to a DNA encoding a bait protein (i.e., protein A described above) such as a ligand binding region of a ligand-responsive transcriptional regulator, and then linked to the downstream of a promoter capable of functioning in host cells. On the other hand, usable examples of the transcriptional activation region of a transcriptional activator include a GAL4 transcriptional activation region, an Escherichia coli-derived B42 acid transcriptional activation region, a herpes simple virus VP16 transcriptional activation region, and the like. A DNA encoding such a transcriptional activation region is linked to a DNA encoding a prey protein (i.e., protein B described above), and then linked to the downstream of the promoter capable of functioning in host cells.
(203) Specifically, examples of the vector that has a DNA encoding a DNA binding region of transcriptional regulator GAL4 and that can use a budding yeast as a host cells include plasmid pGBT9 (produced by Clontech), etc. Examples of the vector that has a DNA encoding a GAL4 transcriptional activation region and that can be used in budding yeast include plasmid pGAD424 (produced by Clontech), etc. Examples of the vector that has a DNA encoding a GAL4 DNA binding region and that can be used in mammalian cells include pM (produced by Clontech), pBIND (produced by Promega), etc. Examples of the vector that has a DNA encoding a simple herpes virus VP16 transcriptional activation region and that can be used in mammalian cells include pVPl6 (produced by Clontech), pACT (produced by Promega), etc. Examples of the vector that has a DNA encoding a LexA DNA binding region and that can be used in mammalian cells include pLesA (produced by Clontech), etc. Examples of the vector that has a DNA encoding B42 and that can be used in mammalian cells include pB42AD (produced by Clontech), etc.
(204) In this case, for example, a vector in which the ALuc gene of the present invention is inserted as a reporter gene into the downstream of the region (e.g., USAG) to which GAL4 binds may be formed. In the case of mammalian hosts, by using a commercially available pG5Luc vector (Promega) or pFR-Luc vector (Stratagene), the luciferase (ALuc) of the present invention can be easily used by a known method in place of firefly luciferase incorporated into the vector. The luciferase (ALuc) of the present invention can also be used in place of chloramphenicol acetyltransferase (CAT) of a commercially available pG5CAT vector (Clontech).
(205) (5-4) Activatable Method
(206) The analysis system carrying a bioluminescent enzyme as a reporter protein according to the activatable method has been also studied and developed by the present inventors as a bioluminescent probe technique. Examples of application of the ALuc of the present invention to a bioluminescent probe and an intracellular imaging method using the bioluminescent probe are explained below as typical examples of the activatable method. Before this explanation, the luminescent fusion protein (luminescent capsule) developed for the first time in the present invention is explained. In addition, the ALuc of the present invention can be suitably used as a reporter protein used in protein complementation assays (PCA) and protein splicing assays (PSA), which are included in the activatable method.
(207) (i) Production of Luminescent Fusion Protein (Luminescent Capsule)
(208) By binding a membrane localization signal to the C-terminus of the ALuc of the present invention, the ALuc can be localized in the plasmamembrane. Such a molecular design allows smooth supply of the substrate and oxygen, enabling visualization of stable bioluminescence with extremely high intensity. For the visualization, it is possible to insert a polypeptide or protein gene as a cargo between the ALuc and a nucleic acid encoding the signal peptide. This allows efficient transfer of the cargo protein to the plasma membrane surface, and makes the place where the protein is transferred illuminated. One typical example is as follows. When the DEVD sequence or IETD sequence responsive to cell death signal is inserted between proteins, the DEVD sequence or IETD sequenceactively responds to the activities of caspase-3 or caspase-8 as signals at the cell death, and functions as a visualization system. The present inventors name the luminescent fusion protein with this structure a luminescent capsule.
(209) Compared to conventional luminescent probes, the luminescent capsule shows stable optical properties with remarkably high intensity, and is responsive to a specimen that cannot pass through the plasma membrane. The luminescent capsule has a structure in which a membrane localization signal (MLS) is linked to the C-terminus of the luminescent enzyme as a basic frame structure. Since the effect of a compound causing a form change on the cell surface, such as a compound inducing cell death, can be visualized as a form change in the plasma membrane surface, by this structure or even when the luminescent enzyme of the present invention is linked to a tandem to enhance the amount of luminescence, easy observation is possible. Preferably, it is possible to insert between the MLS and the C-terminus of the luminescent enzyme, a polypeptide causing a form change in the plasma membrane surface, or the partial recognition sequence of the peptide, specifically, the full length or the partial recognition sequence of a G-protein coupled receptor (GPCR) or c-SRC. Further, by inserting a polypeptide inducing cell death or the recognition sequence of the peptide as a cargo between the MLS and C-terminus of the luminescent enzyme, cell death can be visualized. More specifically, when a peptide sequence (generally 20 amino acids or less, preferably 10 amino acids or less) recognized by caspases, proteases (e.g., serine protease and cystein protease), or digestive enzymes (e.g., trypsin and amylase), for example, an amino acid sequence containing DEVE or IETD used in Example 1-7 is inserted as a cargo, cell death can be visualized by caspase-3 activities. Further, by linking a fluorescence protein or another luminescent enzyme as a cargo between the luminescent enzyme and MLS, the amount of luminescence on the plasma membrane surface is increased as in the case where the luminescent enzyme of the present invention is linked to a tandem, allowing easy observation of the plasma membrane form. Since this fusion protein even responds to a ligand that cannot pass through the plasma membrane, screening with respect to various stimulations is possible.
(210) The luminescent capsule of the present invention is a luminescent fusion protein in which a protein or polypeptide, which is intended to be expressed on the plasma membrane surface, is inserted between the membrane localization signal (MLS) and the C-terminus of the ALuc of the present invention. Typical examples include
(211) (a) a luminescent fusion protein wherein a fluorescence protein or luciferase is inserted between the membrane localization signal (MLS) and the C-terminus of the ALuc of the present invention (the luciferase may be ALuc other than the present invention), and
(b) a luminescent fusion protein wherein a polypeptide changing the form in the plasma membrane, or a polypeptide having 20 or less amino acids, preferably 10 or less amino acids recognized by the polypeptide changing the form in the plasma membrane, is inserted between the membrane localization signal (MLS) and the C-terminus of the ALuc of the present invention. The polypeptide changing the form in the plasma membrane is particularly preferably a polypeptide inducing cell death, and more preferably a polypeptide having 20 or less amino acids containing caspase or the recognition sequence of the caspase, i.e., DEVD or IETD.
(ii) Application to Luminescent Probe
(212) Further, by incorporating the ALuc of the present invention into the integrated-molecule-format luminescent probe (Non-patent Literature 4, Non-patent Literature 6, Non-patent Literature 9, Non-patent Literature 10, Patent Literature 1 to 4) or the two-molecule-format luminescent probe (Non-patent Literature 5 and Non-patent Literature 8), which are recited in the pending patents applied by the present inventors, the presence or absence of ligand and the intensity of ligand activity can be observed with high luminance. By comprising, as the probe components, (i) the bisected luminescent enzyme (N- and C-terminal fragments), and (ii) a ligand-binding protein responsive to the target ligand and (iii) a recognition protein that recognizes the bond of the ligand with the ligand-binding protein, which are linked to the vicinity of the bisected luminescent enzyme, it is possible to form a high-performance luminescent probe. This luminescent probe functions such that, as the recognition protein recognizes the ligand binding of the ligand-binding protein, the two adjacent fragments of the bisected enzyme complement each other and thereby change the enzymatic activity. Here, due to the high luminescence intensity and stability of the bisected enzyme, it is possible to perform reliable measurement with an improved detection limit.
(213) In the present invention, integrated molecule-format luminescent probe denotes a known bioluminescent probe in which all components for visualization imaging are integrated in a single fusion molecule (disclosed in Patent Literature 1-2). For example, integrated molecule-format luminescent probe denotes a fusion protein that comprises, as fundamental components, the two fragments of N- and C-terminals obtained by bisecting the ALuc of the present invention, a ligand-binding protein, and a recognition protein for recognizing the ligand-binding protein. Similarly, two molecule-format luminescent probe in the present invention denotes a bioluminescent probe in which the two fragments of N- and C-termini obtained by bisecting the ALuc of the present invention are present in the fusion protein containing the ligand-binding protein, and in the fusion protein containing the recognition protein, respectively (see Example 1-10 of the present invention).
(214) When the ALuc of the present invention is used for these bioluminescent probes, the ALuc must be bisected into an N-terminal fragment and a C-terminal fragment. The bisected portion is the same as the bisected portion shown in Example 1-10 or corresponding portions of other ALucs.
(215) Patent Literature 1 to 4 disclose the details regarding the actual method for using the superluminescent enzyme of the present invention as an integrated molecule-format luminescent probe. More specifically, the luciferase (ALuc) of the present invention is bisected, and a chimera DNA encoding a luminescent probe in which a ligand-binding protein and a peptide sequence, which recognizes the change in steric structure upon binding of a ligand to the protein, are linked in a linear chain form. Generally, the chimera DNA is subcloned into a vector suitable for the cells in which the chimera DNA is intended to be expressed, and the vector is introduced into the cells to be expressed. However, the chimera DNA may be ligated to a control sequence at an upstream portion to be directly introduced into the cells. The target cells are preferably mammalian-derived cells, such as human cells. Other suitable examples include cells that exist in a living subject, and culture cells that retain the native function, yeast cells, insect cells, and prokaryotic cells such as Escherichia coli. The type of the vector is also not particularly limited. A suitable vector capable of being expressed in the target host cells is appropriately selected. The introduction of the vector into the cells is performed using known transfection methods such as a microinjection method or an electroporation method, or a transfection method using a lipid (BioPORTER (Gene Therapy Systems, Inc.), Chariot (Active Motif), etc.).
(216) Since the bioluminescent probe using the superluminescent enzyme of the present invention is introduced into cells as a chimera DNA and expressed in the cells as a fusion protein, by measuring the change in light amount emitted from the cells after subjecting the transformed cell to ligand stimulation, the property or levels of activity of the ligand may be evaluated.
(217) When the superluminescent luciferase (ALuc) of the present invention is incorporated in the bioluminescent probe, the ligand-binding protein, which can be incorporated in the probe together with the ALuc, is intended to mean a protein that binds with a ligand at the ligand binding site. The ligand-binding protein may serve to, in response to the bond with the ligand, for example, change the steric structure, cause phosphorylation, or facilitate protein-protein interaction. Examples of such ligand-binding proteins include nuclear receptors (NR) to which such ligands as hormones, chemical substances, or signal transduction proteins bind; cytokine receptors; and various protein kinases. A suitable ligand-binding protein is selected depending on the target ligand. The ligand that binds to the ligand-binding protein is not particularly limited insofar as it binds to the ligand-binding protein. The ligand may be an extracellular ligand that is introduced in response to extracellular stimulus, or an intracellular ligand that is produced inside the cells. Examples thereof include agonists or antagonists of the receptor protein (for example, intranuclear receptor, or G-protein-linked receptor), signal transduction proteins such as cytokine, chemokine, or insulin, intracellular second messenger, lipid second messenger, phosphorylated amino acid residue, G-protein-linked receptor ligand, and like ligands that specifically bind to proteins involved in intracellular signal transduction.
(218) For example, when the intracellular second messenger, the lipid second messenger, or the like is used as a ligand, the binding domain of each second messenger may be used as the ligand-binding protein. Second messenger denotes a different kind of intracellular signal transduction substance that is newly produced as a result of the bond of the extracellular signal transduction substance, such as a hormone or neurotransmission substance, with a receptor that exists in the plasma membrane. Examples of the second messengers include cGMP, AMP, PIP, PIP.sub.2, PIP.sub.3, inositol trisphosphate (IP.sub.3), IP.sub.4, Ca.sup.2+, diacylglycerol, and arachidonic acid. For example, for Ca.sup.2+ as the second messenger, calmodulin (CaM) may be used as the ligand-binding protein.
(219) (iii) Intracellular Imaging
(220) Further, using the gene encoding the ALuc enables stable introduction of the ALuc into various cell strains. For example, using the gene enables stable introduction of the ALuc into the undifferentiated embryonic cells, ES cells, novel induced pluripotent stem cells (iPS cells). Since the cells do not emit light themselves, it has been very difficult to research the intracellular molecular phenomenon and tissue specificity of the cells. To address this difficulty, a molecular probe containing the ALuc is introduced into somatic cells before the embryo is formed, and then the embryo is differentiated into various tissues. This enables measurement of specific molecular phenomena in respective organs with high sensitivity.
(221) This process is performed according to the method of Yamanaka et al. (Non-patent Literature 17).
(222) Further, by linking the ALuc of the present invention to a suitable signal peptide, the ALuc can be used for luminance imaging of various organelles. For example, by linking a GAP-43-derived MLCCMRRTKQV sequence (SEQ ID NO: 1) to an N- or C-terminus of ALuc, the ALuc may be localized in the plasma membrane. Linking a GRKKRRQRRR sequence (SEQ ID NO: 2) to a terminus enables localization in cell cytoplasm. Further, for localization in endoplasmic reticulum (ER) and cellular nucleus, KDEL (SEQ ID NO: 3) and DPKKKRKV (SEQ ID NO: 4) sequences, respectively, are linked to a terminus. Furthermore, by linking to HIS-tag (HHHHHH) (SEQ ID NO: 5), FLAG-tag (DYKDDDDK) (SEQ ID NO: 6), Myc-tag (EQKLISEEDL) (SEQ ID NO: 7), HA-tag (YPYDVPDYA) (SEQ ID NO: 8), V5-tag (GKPIPNPLLGLDST) (SEQ ID NO: 9), T7-tag (MASMTGGQQMG) (SEQ ID NO: 10) or like antigen sites, the ALuc can be used for immunostaining or separation/refinement in acellular systems. In these usages, known immunostaining technologies or immunocytochemistry may be adopted.
[II] DETERMINATION OF OPTIMUM REACTION SOLUTION FOR BIOASSAY
1. Buffer for Bioassay
(223) (1-1) Lysis Buffer (Cell Lysis Solution) and Assay Buffer (Reaction Solution)
(224) Conventionally conducted bioassays involve two separate assay buffers: a buffer for lysis (cell lysis solution); and a buffer for assay (reaction solution). This is because high lytic activity and low inhibitory effect on a luminescent enzyme are considered essential for quick lysis of the cells, whereas stable assay conditions and removal or analysis of self-luminescence inducing components to reduce background are considered essential for a bioassay reaction.
(225) Promega Corporation has been selling a lysis buffer and an assay buffer under the respective trade names of Luciferase Lysis Buffer (catalog number: E291A) and Luciferase Assay Buffer (catalog number: E290A). New England Biolabs Inc. (NEB) has also been selling a lysis buffer and an assay buffer under the respective trade names of Luciferase Lysis Buffer (catalog number: B3321) and Luciferase Assay Buffer (catalog number: E3300S). Although neither Promega nor NEB discloses the formulations of their commercial products, both disclose complex protocols in which a lysis buffer and an assay buffer are separately used.
(226) In the present invention, as stated above, studies on the buffer component formulations shown below were conducted with the intent of simplifying the complex protocols, and enhancing the reaction stability and the sensitivity.
(227) (a) surfactant: polyoxyethylene octylphenyl ether (Triton X-100; TX100), Nonidet P-40 (NP40), polyoxyethylene sorbitan monolaurate (Tween20; TW20), polyoxyethylene sorbitan monooleate (TW80), polyoxyethylene cetyl ether (Brij58), sodium dodecyl sulfate (SDS), and the like. The degree of hydrophilicity is indicated as TW20>Brij58>TW80>TX100>NP40; and the degree of the power of surfactant is indicated as NP40>TX100>Brij58>TW20>TW80.
(b) salts: NaCl, KCl, (NH.sub.4).sub.2SO.sub.4, and the like
(c) SH reagents: mercaptoethanol, DTT, and the like
(d) polyols: glycerol, glucose, sucrose, and the like
(e) glycols: polyethylene glycol (PEG), polypropylene glycol (PPG)
(f) chelate reagents: EGTA, EDTA, and the like
(g) protease inhibitors: aprotinin (molecular weight: 6.5 kD), leupeptin (molecular weight: 427), pepstatin A (pepstatin, molecular weight: 686), phenylmethylsulfonyl fluoride (PMSF, molecular weight: 174), antipain (antipain, molecular weight: 605), chymostatin (chymostatin, molecular weight: 608), pefabloc SC (AEBSF, 240 Da), DFP (184 Da), p-APMSF (216 Da), STI (20,100 Da), leupeptin (460 Da), N-tosyl-L-phenylalaninechloromethylketone, 3,4-dichloroisocoumarin (215 Da), EDTA-Na.sub.2 (372 Da), EGTA (380 Da), 1,10-phenanthroline (198 Da), phosphoramidon (580 Da), dithiobis (2-amino-4-methylpentane), E-64 (357 Da), cystatin, bestatin, epibestatin hydrochloride, aprotinin, minocycline, ALLN (384 Da), and the like
(g) buffer agents: p-toluenesulfonic acid, tartaric acid, citric acid, phthalate, glycine, trans-aconitic acid, formic acid, 3,3-dimethylglutaric acid, phenylacetic acid, sodium acetate, succinic acid, sodium cacodylate, sodium hydrogen maleate, maleic acid, sodium phosphate, KH.sub.2PO.sub.4, imidazole, 2,4,6-trimethylpyridine, triethanolamine hydrochloride, sodium 5,5-diethylbarbiturate, N-ethylmorpholine, sodium pyrophosphate, tris(hydroxymethyl)aminomethane, bicine, 2-amino-2-methylpropane-1,3-diol, diethanolamine, potassium p-phenolsulfonate, boric acid, sodium borate, ammonia, glycine (glycine), Na.sub.2CO.sub.3/NaHCO.sub.3, sodium borate, or a combination thereof
(h) Others: sodium molybdate (stabilization of receptors), dithiothreitol (dithiothreitol, DTT) (reducing agent)
(1-2) Buffer Component 1, a Basic Buffer, of the Present Invention (HBSS Buffer)
(228) In the present invention, the HBSS buffer (Hanks' balanced salt solution) is used as a basic composition. An HBSS buffer was prepared in accordance with a known protocol (e.g., see the website of National Institute of Biomedical Innovation at http://cellbank.nibio.go.jp/legacy/sheet/att00011.htm), as described below.
(229) First, the following four types of solutions are prepared beforehand, and mixed for use.
(230) Solution 1: 1.4% NaHCO.sub.3 solution
(231) Solution 2: a solution prepared by dissolving 80.0 g of NaCl, 4.0 g of KCl, 2.0 g of MgSO.sub.4.7H.sub.2O, 0.6 g of Na.sub.2HPO.sub.4.2H.sub.2O, 10.0 g of glucose, and 0.6 g of KH.sub.2PO.sub.4 in 800 ml of water
(232) Solution 3: a solution prepared by dissolving 1.4 g of CaCl.sub.2 in 100 ml of water
(233) Solution 4: a solution prepared by weighing 0.4 g of phenol red, making it into a paste with a small amount of water, and adding water thereto to give 150 ml of a solution
(234) The mixture is adjusted to a pH of 7.0 with a sodium hydroxide solution (N/20) so as to give 200 ml.
(235) For use, 2.5 ml of solution 1, 8 ml of solution 2, 1 ml of solution 3, and 1 ml of solution 4 are added to 87.5 ml of sterile water. When phenol red is not necessary, solution 4 can be omitted.
(236) (1-3) Buffer Component 2, a Basic Buffer, of the Present Invention (Tris-Buffer)
(237) The Tris buffer refers to a widely used conventional buffer component (as used herein, tris is an abbreviation for tris(hydroxymethyl)aminomethane, which is typically prepared by adding HCl to 10 mM of a tris salt to thereby adjust the pH, and optionally adding 1 mM of EDTA thereto as an additive), and is used in a variety of biological studies because of its high biocompatibility. Nonetheless, there has been insufficient study of the effects of the Tris buffer on a bioluminescent reaction.
(238) In the present invention, it was found that a Tris buffer can be suitably used for bioluminescence, and can be a basic buffer component usable in both lysis and assay.
(239) (1-4) Buffer Formulation in the Present Invention
(240) The above-stated basic buffer components, an HESS buffer and a Tris-buffer, are combined for use. These buffers are mixed at a ratio of 20 to 50:50 to 20, preferably 40 to 60:60 to 40, and most preferably 60:40 in volume % (v/v).
(241) The surfactants NP-40, TW80, and SDS are combined for use. The NP-40, TW80, and SDS are mixed at a ratio of 1:0.1 to 1:0 to 0.5, preferably 1 to 2:0.5 to 2:0.1 to 1, and most preferably 1:1:0.1 in volume % (v/v).
(242) The surfactant TW80 is mixed with other surfactants and the ratio is adjusted to be 1 to 10 volume % (v/v), and preferably 5 to 10 volume % (v/v).
(243) For polyols, polyethylene glycol (PEG), and a sugar component (sucrose, glucose) are combined. PEG400 is contained in an amount of 0.01 to 10 volume % (v/v), and the sugar component. (sucrose, glucose) is contained in an amount of 0 to 20 mg/mL. PEG400 is preferably contained in an amount of 0.1 to 10 volume % (v/v), and the sugar component (sucrose, glucose) is preferably contained in an amount of 2 to 10 mg/mL.
(244) For heavy metals, Fe(III), Cu(II), Mo(VI), and Zn(II) can be contained singly or in a combination in a concentration within a range of 0.01 to 1 PPM, and preferably 1 PPM.
(245) The halogen ions Br.sup. and I can be contained singly or in combination in a concentration of 1 to 100 mM, and preferably 50 to 100 mM.
(246) It is further preferable to optionally add a reducing agent, such as vitamin C, to improve the luminescence stability.
(247) In the Examples, buffer formulations that showed excellent results in the bioluminescence measurement step after the cell lysis step were basically those for which a C3 buffer was used as a cell lysis solution. The probable reason for the excellent results is that the C3 buffer comprises, in addition to a Tris-HCl buffer as a basic buffer, NP-40 having excellent surfactant power and MgCl2 having high physiological compatibility. The buffer combinations that particularly showed excellent results are as follows:
(248) 1. After cell lysis with a C3 buffer, C8 and C10 assay buffers were used.
(249) 2. After cell lysis with a C3 buffer, a C6 assay buffer was used.
(250) 3. After cell lysis with a C3 buffer, an assay buffer prepared by adding Al(III), Ca(II), Cu(II), Fe(III), or Mg(II) to an HBSS buffer was used.
(251) 4. After cell lysis with a C3 buffer, an assay buffer prepared by adding 1% PEG or PPG to an HBSS buffer was used.
(252) 5. After cell lysis with a C3 buffer, an assay buffer prepared by adding 50 mM of KI or 100 mM of KBr to an HESS buffer was used.
(253) 6. After cell lysis with a C3 buffer, an assay buffer prepared by adding 2 mg/mL of D(+)glucose or glycine to an HBSS buffer was used.
(254) From the above results, preferable buffer formulations as a one-shot reaction solution were narrowed down as shown below.
(255) Specifically, it was found that a basic formulation of one-shot reaction solution in a bioluminescent enzyme utilization technique, where prompt lysis and observation under high luminescent intensity are required, can be established by combining a Tris-HCl buffer, which is a basic buffer of the C3 buffer, with an HESS buffer, and further combining a surfactant, NP-40 or SDS, salts such as Al(III), Ca(II), Cu(II), Fe(III), or Mg(II), PEG or PPG, a halogen ion (I.sup., Br.sup., and D(+)glucose or glycine.
(256) On the basis of such an idea, C14 to C18 buffers basically comprising a combination of a C3 buffer and an HESS buffer were established as a one-shot reaction solution. However, when used for bioluminescence probes, C14 to C18 buffers failed to show preferable results. When C19 to C22 buffers comprising TW80 in place of an HBSS buffer were used, C19, C21, and C22 buffers prepared by combining TW80 with a C3 buffer in an amount of 1 to 10% without adding SDS were found to serve as a one-shot buffer that enables fast measurement. TW80 was selected as an additional additive because TW80 can balance with NP-40 in terms of surfactant hydrophilicity and the power of surfactant.
(257) The present inventors had previously found in experiments conducted using another luminescent enzyme that a Tris-HCl buffer, when comprising the surfactant NP-40 in combination with SDS, increases luminescence intensity (the results not shown). Thus, the present inventors conducted the following experiment using a C4 buffer comprising SDS in addition to a Tris-HCl buffer and NP-40, in place of a C3 buffer comprising a Tris-HCl buffer and NP-40 as basic components. More specifically, the present inventors conducted the experiments using, as a one-shot buffer, C23 to C26 buffers each comprising, in addition to a C4 buffer as a basic component, TW80 and an HBSS buffer in a different formulation. The present inventors found that C23, C24, and C25 enable fast measurement with high S/N ratios.
(258) As described above, one-shot reaction buffers for a bioluminescent enzyme were established by combining a C4 buffer with a C13 buffer comprising an HBSS buffer as a basic component, and adding TW80 and D-luciferin as a substrate thereto.
(259) (1-5) Buffer Solution Used in the Examples of the Present Invention
(260) The following shows the formulations of the buffer solutions used in the Examples of the present invention.
(261) TABLE-US-00001 TABLE 1 Buffer Formulations Name (Abbrev. Name) Buffer Basic Substance Additive 1 Additive 2 Additive 3 Additive 4 Composition 1 (C1) 20 mM Tris-HCl (pH 7.5) 1% Triton 1 mM EDTA 1 mM Na.sub.3VO4 150 mM NaCl 2.5 mM Sodium Pyrophosphate 1 mM EGTA 1 g/ml 1 mM Glycerophosphate Leupeptin Composition 2 (C2) 50 mM Tris-HCl (pH 6.8) 1% (w/v) SDS 10% (v/v) 10% (v/v) 0.001% (w/v) 2-Mercaptoethanol Glycerol Bromophenol Blue Composition 3 (C3) 20 mM Tris-HCl (pH 7.4) 0.05% (w/v) 0.05% Sodium 2.5 mM Magnesium 200 mM NaCl NP-40 Azide Chloride Composition 4 (C4) 25 mM Tris-HCl (pH 7.6) 1% NP-40 1% Sodium 150 mM NaCl 0.1% SDS Deoxycholate Composition 5 (C5) H.sub.2O None None None Composition 6 (C6) Sodium Phosphate Buffer (PBS) 145 mM NaCl Composition 7 (C7) Sodium Phosphate Buffer (PBS) 145 mM NaCl 0.5% BSA Composition 8 (C8) HBSS buffer Composition 9 (C9) Tris buffer EDTA Composition 10 (C10) Tris buffer EDTA Polyethylene Glycol (PEG) Composition 11 (C11) Tris buffer 10 mM MgCl.sub.2 Composition 12 (C12) Tris buffer 50 mM MgCl.sub.2 Composition 13 (C13) HBSS buffer PEG 100 0.01% Fe (III) 0.1 ppM, As (v) 0.1 ppM
(262) TABLE-US-00002 TABLE 2 Buffer Formulations Mixing Ratio of 1.sup.st and Name (Abbrev. Name) 1st Buffer 2nd Buffer Additive 1 Additive 2 2.sup.nd Buffers Composition 14 (C14) Formulation of C3 HBSS Buffer 2:8 Composition 15 (C15) Formulation of C3 HBSS Buffer 4:6 Composition 16 (C16) Formulation of C3 HBSS Buffer 6:4 Composition 17 (C17) Formulation of C3 HBSS Buffer 8:2 Composition 18 (C18) Formulation of C3 HBSS Buffer 10:0 Composition 19 (C19) Formulation of C3 TW80 1% Composition 20 (C20) Formulation of C3 TW80 1% SDS 0.1% Composition 21 (C21) Formulation of C3 TW80 5% Composition 22 (C22) Formulation of C3 TW80 10% Composition 23 (C23) Formulation of C4 + HBSS Buffer 2:8 TW80 1% Composition 24 (C24) Formulation of C4 + HBSS Buffer 4:6 TW80 1% Composition 25 (C25) Formulation of C4 + HBSS Buffer 6:4 TW80 1% Composition 26 (C26) Formulation of C4 + HBSS Buffer 8:2 TW80 1% Composition 27 (C27) Formulation of C4 + Formulation of C13 Compositional Ratio of TW80 1% C4 to C13 = 6:4 Composition 28 (C28) Formulation of C4 + Formulation of C13 D-luciferin Compositional Ratio of TW80 1% C4 to C13 = 6:4 Composition 29 (C29) Formulation of C4 + Formulation of C13 Mg.sup.2+ (1 ppm) Compositional Ratio of TW80 1% D-luciferin C4 to C13 = 6:4
2. Bioassay of Interest in the Present Invention
(263) The present invention relates to a buffer formulation that is expected to be applied to the following bioassays. In conducting a reporter-gene assay, two-hybrid assay, protein complementation assay, intein-mediated protein splicing assay, or single-chain probe-based assay, a measurement is directly carried out with cultured cells without carrying out the cell lysis step.
(264) For example, in a reporter-gene assay, cells transfected with a reporter expression vector in a 96-well plate are ligand-stimulated. After that, when the cells produce luminescence, 50 L of the reaction solution is added, and a measurement is immediately conducted.
(265) When an integrated-molecule-format bioluminescent probe is used, cells for expressing the integrated-molecule-format bioluminescent probe in a 96-well plate are ligand-stimulated. After stimulation, 50 L of the reaction solution is added, and a measurement is immediately conducted.
3. Luminescent Enzyme for Use in Bioassay of the Present Invention
(266) The luminescent enzyme for use in the bioassays of the present invention includes all types of luminescent enzymes. Examples of the luminescent enzyme include bioluminescent enzymes derived from insects and marine animals, typically firefly luciferases, click beetle luciferases, Renilla luciferases, and copepod luciferases (Metridia longa luciferase, Metridia pacifica luciferase).
(267) As used herein, the term copepod luciferases refers to luciferases sharing common enzyme activity and structural characteristics with luciferases originating from known copepods. Specifically, such luciferases are those having an optimum pH of about 5 to 8, an optimum temperature of about 4 to 25 C., and enzyme activity that catalyzes a luminescent reaction with coelenterazine as a substrate. The luciferases comprise two enzyme active domains with a secretion signal at their N-terminus, and have a molecular weight of about 20 kD (18 kD to 28 kD), which is the smallest among all of the luminescent enzymes.
(268) Preferable examples of luminescent enzymes usable in the bioassays of the present invention include the aforementioned novel artificial luciferases (ALuc) according to the present invention.
4. Measuring Procedure and Measuring Apparatus Used in the Present Invention
(269) The ligand activity can be measured in accordance with a typical bioluminescence assay, and conventional protocols can be used without any restriction.
(270) Luminometers (e.g., Mini Lumat LB 9506, Berthold; and GloMax 20/20n, Promega) have typically been used to measure bioluminescence intensity. A cell lysis solution is poured over cultured cells in a plate to thereby produce a cell lysate. After the cell lysate is mixed with a substrate, the luminescence is immediately measured.
(271) To measure the ligand activity of cultured cells in a 96-well plate, a ready-made bioluminescence plate reader (e.g., Mithras LB 940, Berthold; and SH-9000, Corona) can be used. Using a substrate solution autoinjector attached to the plate reader, a substrate can instantaneously be introduced, and bioluminescence generated by the expressed probe can instantaneously be measured in the presence of the ligand.
5. Analyte of Interest in Screening Method
(272) Examples of analytes in these screening methods include organic or inorganic compounds (particularly compounds of low molecular weight), proteins having bioactivity, and peptides. These substances may be those whose function and structure are either known or unknown. A combinatorial chemical library can be an effective means as a group of analytes for efficiently identifying target substances. The preparation and screening of a combinatorial chemical library are well known in the art (see, e.g., U.S. Pat. Nos. 6,004,617 and 5,985,365). Alternatively, a commercially available library may be used (e.g., libraries available from ComGenex (US), Asinex (Russia), Tripos Inc. (US), ChemStar, Ltd. (Russia), 3D Pharmaceuticals (US), and Martek Biosciences). By applying a combinatorial chemical library to a cellular cluster for expressing a probe, a high-throughput screening can be carried out.
6. Kit
(273) The present invention also provides a bioassay kit comprising the aforementioned reaction buffers for bioluminescence. The kit according to the present invention may optionally comprise various components for carrying out a bioassay. Examples of such components include, but are not limited to, luminescent enzymes, vectors comprising genes for encoding luminescent enzymes, cells for expressing luminescent enzymes, luminescent substrates, various instruments (96-well plates, and tubes), and control samples. The kit may also comprise a user manual describing the procedure for carrying out the bioassays according to the present invention.
(274) Preferable examples of luminescent enzymes include bioluminescent enzymes derived from insects and marine animals, typically firefly luciferases, click beetle luciferases, Renilla luciferase, copepod luciferases (Metridia longa luciferase, Metridia pacifica luciferase), and the artificial luciferases (ALuc) described in section [I] above. The artificial luciferases (ALuc) are particularly preferable examples.
(275) A vector comprising a gene for encoding a luminescent enzyme can be produced in accordance with a known technique depending on the intended bioassay (e.g., reporter-gene assay, two-hybrid assay, protein complementation assay, intein-mediated protein splicing assay, and single-chain probe-based assay).
(276) When the luminescent enzyme for use is, for example, a copepod luciferase or the artificial luciferases (ALuc) of the present invention, a preferable luminescent substrate is coelenterazine, which refers to both native coelenterazine (native CTZ) and the derivatives of native coelenterazine.
(277) Examples of control samples include positive controls comprising a luminescent enzyme in a predetermined amount, and negative controls not comprising a luminescent enzyme.
(278) The kit according to the present invention can be produced by combining the above-described components in accordance with a known technique. The kit according to the present invention can be used for carrying out the aforementioned bioassays of the present invention.
[III] TERMS AND CONCEPTS USED IN THE PRESENT INVENTION
(279) The other terms and concepts used in the present invention are specifically defined in the descriptions of embodiments and examples of the invention. The terms are generally selected from the IUPAC-IUB Commission on Biochemical Nomenclature, or based on interpretations of idiomatic terms and words in the related field. Except for the techniques with apparent sources, the various techniques used to carry out the present invention can be easily and consistently performed by one of ordinary skill in the art with reference to published documents, etc. For example, genetic engineering and molecular biological techniques can be carried out according to J. Sambrook, E. F. Fritsch & T. Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); D. M. Glover et al. ed., DNA Cloning, 2nd ed., Vols. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1995; Japanese Biochemical Society ed., Zoku Seikagaku Jikken Koza 1 [Continuation of Biochemistry Experimental Series 1], Idensi Kenkyu Ho [Gene Study Method] II, Tokyo Kagaku Dojin (1986); Japanese Biochemical Society ed., Shin Seikagaku Jikken Koza 2 [New Biochemistry Experimental Series 2], Kakusan [Nucleic Acid] III (Kumikae DNA Gijutsu [DNA Recombinant Technology]), Tokyo Kagaku Dojin (1992); R. Wu ed., Methods in Enzymology, Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. ed., Methods in Enzymology, Vols. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. ed., Methods in Enzymology, Vols. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E), & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), etc.; the methods mentioned in the documents referenced in these documents; or other various similar methods and modified methods thereof that are substantially the same as the disclosed methods. The proteins, peptides, and DNAs encoding them used in the present invention are available from existing databases (e.g., URL: http://www.ncbi.nlm.nih.gov).
EXAMPLES
(280) The following examples specifically describe the present invention in more detail; however, the present invention is not limited to the Examples.
(281) The other terms and concepts used in the present invention are based on the interpretations of idiomatic terms and words in the related field. Except for the techniques with apparent sources, the various techniques used to carry out the present invention can be easily and consistently performed by one of ordinary skill in the art with reference to published documents, etc. The various analyses were performed in accordance with the methods disclosed in instruction manuals, catalogs, or the like of the analytical instruments, reagents, and kits used in the analyses.
(282) The disclosures of the technical documents, patent publications, and specifications of pending patent applications cited herein are incorporated into the present specification by reference.
(Example 1-1) Extraction of Amino Acid Sequences of Artificial Luciferases (ALucs)
(283) According to the publicly known database of the National Center for Biotechnology Information (NCBI), etc., copepod luciferase sequences were aligned based on the similarity of amino acids to find frequently occurring amino acids. Based on the frequently occurring amino acids, novel artificial luciferase (ALuc) prototypes were made (
(284) Based on the many artificial luciferase (ALuc) sequences obtained from the similarity alignment above, gene codons optimized for mouse cells were suitably applied to construct the same number of artificial gene sequences. Based on these gene sequences, actual artificial genes were obtained from an outsourcing company (Operon Biotechnology, Co., Ltd.) specializing in artificial gene synthesis.
(Example 1-2) Comparison of Luminescence Intensity, Luminescence Stability, and Red-Shifted Degree of Artificial Luciferases (ALucs)
(285) The artificial luciferase (ALuc) genes synthesized by the above process were subcloned into mammalian cell expression vectors (pcDNA3.1 (+)). Each expression vector was transfected with African green monkey kidney-derived COS-7 cells, and the luminescence intensity, luminescence stability, and red-shifted degree of artificial luciferases (ALucs) were compared (
(286) First, the luminescence intensity of artificial luciferases (ALucs) was compared (
(287) The results indicated that ALuc2, ALuc9, ALuc10, ALuc15, ALuc16, ALuc18, ALuc22, ALuc23, ALuc25, etc. exhibited relatively high luminescence intensity. According to the luminescence intensity measurement by using the luminescence image analyzer, these ALucs were confirmed to have luminescence intensity about 50 folds higher than that of conventional Gaussia-derived luciferase (GLuc), Renilla reniformis-derived luciferase (Renilla luciferase), and Metridia pacifica-derived luciferase (MpLuc4).
(Example 1-3) Examination of Luminescence Stability of Artificial Luciferases (ALucs)
(288) The luminescence stability of the artificial luciferases (ALucs) of the present invention was examined in various respects (
(289) This experiment was expanded to artificial luciferases ALuc16 to ALuc25 (
(Example 1-4) Heat Resistance and Degree of Extracellular Secretion of Artificial Luciferases (ALucs)
(290) The heat resistance and degree of extracellular secretion of artificial luciferases of the present invention were measured (
(291) The results confirmed that the luminescence intensity of ALuc22 was reduced by about 40% by heating. In contrast, in other luciferases (ALuc16, ALuc23, and ALuc25), a remarkable reduction in luminescence intensity that may be due to heating was not observed.
(292) Further, each luciferase (ALuc) was introduced into COS-7 cells, culturing was performed overnight, and then the amount of each luciferase (ALuc) secreted from the cells was measured based on the luminescence intensity of the medium (
(Example 1-5) Red-Shifted Degree of Bioluminescence of Artificial Luciferases (ALucs) of the Present Invention
(293) The red-shifted degree of bioluminescence was measured based on the luminescence spectrum of each artificial luciferase (ALuc) of the present invention. First, each artificial luciferase (ALuc) was introduced into COS-7 cells, followed by culturing overnight. Subsequently, the cells were treated with a lysate produced by Promega for 20 minutes, and immediately after the introduction of a substrate-containing reaction solution produced by Promega to the 5 L (
(294) The results indicated that when the ALuc of the present invention was used in bioimaging, it exhibited tissue transmittance with extremely high signal bioluminescence.
(Example 1-6) Correlation and Similarity Between Artificial Luciferases (ALucs) of the Present Invention and Luciferases Derived from Known Luminescent Organisms
(295) The correlation and similarity between the amino acid sequences of the artificial luciferases (ALucs) newly synthesized in Examples 1-1 to 1-5 and the amino acid sequences of luciferases derived from known luminescent organisms were compared (
(296) First, the similarity between the ALucs of the present inventions and other luminescence enzymes was examined by using CLUSTALW2.1. The results indicated that the closest luciferase was MpLuc1, and MoLuc1 and MLuc also hit. According to the protein sequence comparison of NCBI Blast, ALuc23 had 83% similarity with MpLuc1, and 74% similarity with MoLuc1. The similar amino acid similarity measurement indicated that ALuc25 had the highest similarity with MpLuc1, and the homology was 72%.
(Example 1-7) Construction of Luminescent Capsule Probe Containing Artificial Luciferase (ALuc) of the Present Invention in its Frame Structures
(297) A luminescent capsule probe containing the artificial luciferase (ALuc) of the present invention in its frame structure was developed (
(298) The luminescent capsule has the ability to localize a fluorescence protein (mPlum), other luciferases (RLuc8.6-535), and a peptide (DEVD sequence, etc.) in the plasma membrane. The luminescence capsule containing fluorescent protein mPlum or peptide DEVD (substrate of caspase-3) was confirmed to exhibit excellent luminescence stability (
(Example 1-8) Effect of Luminescent Capsule of the Present Invention
(299) Effects of the luminescent capsule shown in Example 1-7 were evaluated by using a fluorescence microscope (Leica) (
(Example 1-9) Two-Hybrid Assay Using Artificial Luciferase (ALuc) of the Present Invention
(300) To demonstrate advantages of the artificial luciferase (ALuc) of the present invention as a luminescence reporter, ALuc was used as a reporter for a conventional mammalian two-hybrid assay system. First, using a known gene engineering technique, a novel reporter expression vector obtained by inserting a gene encoding MpLuc4 or ALuc16 into a commercially available reporter expression vector (pG5) was constructed. In addition, pG5-GLuc, which is the result of the researchers' previous study, was also prepared (
(301) In addition to any one of the above three types of reporter expression vectors, a vector (pACT-MyoD) expressing a muscle regulatory factor (MyoD) and a vector (pBIND-ID) expressing a transcriptional regulator (ID) were cotransfected into COS-7 cells. After culturing overnight in a cell culturing device, a lysis solution and a substrate solution produced by Promega were used to cause a luminescent reaction, and difference in luminescence intensity varying according to the reporter difference was compared. The results of the comparison under the same conditions showed that the strongest bioluminescence was observed from the lysate containing the ALuc expression vector (
(302) Along with the measurement, the luminescence value from the supernatant (medium) during cell culturing was simultaneously measured. As a result, slightly strong bioluminescence was observed when ALuc16 or GLuc was introduced. The results indicated that part of the luciferase was secreted out of the plasma membrane.
(Example 1-10) Development of Integrated-Molecule-Format Bioluminescent Probe Containing ALuc in its Frame Structure
(303) To demonstrate the advantages of ALuc, a series of integrated-molecule-format bioluminescent probes containing ALuc in its frame structure was developed (
(304) In the bisection of ALuc16, the probes were named as cSimgr8 (SEQ ID NO: 21) to cSimgr14 according to its bisection point. The bisection point corresponding to each plasmid is as follows: cSimgr8 (125/126), cSimgr9 (129/130), cSimgr10 (133/134), cSimgr11 (137/138), cSimgr12 (137/138; containing a mutant at the joint portion), cSimgr13 (141/142), and cSimgr14 (146/147).
(305) The results indicated that cSimgr13 and cSimgr14 showed good absolute luminescence intensity, and cSingr8 showed a good S/N ratio.
(Example 1-11) Measurement of Toxicity of Cytotoxic Substance (STS) Using Luminescence Measurement Device of the Present Invention
(306) A novel bioluminescence measurement device was developed for conducting a cytotoxicity detecting experiment (
(307) First, a microslide holder was treated with aluminum so that a commercially available microslide for cell culturing or for microscope observation (6-channel, produced by ibidi) fitted the holder. A platform was formed using, for example, a plastic material, so as to engage the microslide holder. Grooves were made in the sides of the slide holder or under the slide holder at certain intervals so that the microslide holder was nonslip on the platform at each interval. The platform was provided with engaging materials (e.g., spring balls) so that the platform engaged the grooves.
(308) By covering the upper portion of the microslide for cell culturing with a honeycomb cap, optical interference between the channels of the microslide was blocked. The surface of the cap was made reflective to reflect light. The shape of the cap is not limited to a honeycomb shape as long as it can block optical interference between the channels of the microslide.
(309) A filter holder (made of metal or the like) for including three optical filters was attached under the platform, and configured so that the slide filter holder was nonslip at the positions corresponding to each of the three optical filters.
(310) A stand made of metal or the like was formed under the filter holder to withstand the weight in the upper portion. A hole was made in the stand, and the surface of the stand was made reflective by plating so that luminescence from the upper portion can reach the lower detection portion without optical loss. By attaching the stand of the luminescence measurement device of the present invention in place of the dish to a luminometer or a spectrophotometer, microslide measurement using a conventional luminometer or spectrophotometer can be easily performed.
(311) Although a 6-channel microslide was used in
(312) Using the luminescence measurement device, the toxicity of a cell death-inducing chemical substance (STS) was measured (see
(313) The luminescence measurement was performed in the following manner. The microslide was washed once with an HBSS buffer, and a substrate-containing HBSS buffer (100 L) was simultaneously introduced into each microslide channel, and the microslide was immediately fixed to the luminescent measurement device. After the device was covered with a mirror cap, the device was mounted on a conventional luminometer (GloMax 20/20n; Promega). The luminescence value was measured by changing the channels and the filters (three-second light condensation, n=3, see
(314) The luminescence probe localized in the membrane was decomposed by Caspase-3 activated by STS to enhance luminescence intensity (
(Example 1-12) Measurement of Toxicity of Cell Death-Inducing Chemical Substance (STS) According to Change in Luminescence Spectrum Using Luminescence Measurement Device of the Present Invention (Measurement 1)
(315) Using the luminescence measurement device of the present invention, the toxicity of a cell death-inducing chemical substance (STS) was measured based on changes in luminescence spectrum (see
(316) The luminescence measurement was performed in the following manner. The microslide was washed once with an HESS buffer, and a substrate-containing HBSS buffer (100 L) was simultaneously introduced into each slide channel, and the microslide was mounted on the measurement device. After the device was covered with a mirror cap, the device was further mounted on a conventional spectrum meter (AB-1850; ATTO) (
(317) Consequently, an increase in the luminescence intensity in the full wavelength was observed in the case with STS (1 M) stimulation compared to the case without STS stimulation. The maximum luminescence wavelength (.sub.max) of the spectrum was 580 nm. About 26% of the total photons were photons greater than 600 nm, which corresponds to a so-called optical window. The results indicated that the use of the probe or device in animal imaging can attain excellent tissue transmittance.
(Example 1-13) Cytotoxic Measurement Using Bioluminescent Capsule Containing ALuc16 in its Frame Structure (Measurement Example 2)
(318) The cytotoxicity of a chemical substance was measured using the bioluminescent capsule (SEQ ID NO: 19) containing ALuc 16 in its frame structure (
(319) Consequently, the luminescence intensity of the right two channels was two folds higher than that of the left two channels. The results indicated that the luminescent capsule of the present invention can measure cytotoxicity with high sensitivity.
(Example 1-14) Measurement of Stress Hormone
(320) Stress hormone was measured using the luminescence measurement device of the present invention (see
(321) As a result, even when the same samples were used, an increase in luminescent value, i.e., from 28% (with stress hormone) to 42% (without stress hormone) was observed in the presence of the mirror cap, as compared with the case in the absence of the mirror cap (
(322) In the stress hormone measurement using the same luminescence samples, the result analysis of
(Example 1-15) Novel Single-Chain Antibody (scFv-ALuc16) Linked with Artificial Luciferase (ALuc)
(323) To demonstrate advantages of the artificial luciferase (ALuc) of the present invention as a luminescence pigment, a novel single-chain antibody (single-chain variable fragment; scFv) in which ALuc was linked with a GST tag antibody variable region fragment was experimentally produced (
(324) This trial product was obtained by linking the GST tag antibody variable region fragment with ALuc16 via a GGGGS linker using a gene engineering technique, and inserting the resultant into an Escherichia coli expression vector (
(325) An anti-mouse antibody (anti-mouse IgG; GE Healthcare) linked with commercially available horseradish peroxidase (HRP) was purchased, and prepared in a manner such that the concentration of HRP was 1 g/mL. scFv-ALuc16 in the same amount as the HRP-linked anti-mouse antibody was prepared, and the solutions were individually injected, 10 L per well, into the wells of a 96-well microplate.
(326) Commercially available ImmunoStar LD (Wako) was prepared as the substrate solution for the HRP-linked anti-mouse antibody, while the substrate solution for scFv-ALuc16a was prepared using a Renilla luciferase assay kit produced by Promega. 90 L per each of the prepared substrate solutions were simultaneously injected into a 96-well microplate using an eight-channel micropipette. The luminescence intensity and temporal change were measured using a luminescence image analyzer (LAS-4000, FujiFilm).
(327) The results indicated that the HRP-linked anti-mouse antibody exhibited about 30% higher luminescence intensity (
(Example 1-16) Luminescence Spectrum Comparison Between Novel Single-Chain Antibody (scFv-ALuc16) and Conventional HRP
(328) The following spectrum measurement was conducted to compare the luminescence spectra between scFv-ALuc16 and conventional HRP (
(329) The results indicated that scFv-ALuc16 showed a more red-shifted luminescence spectrum than the HRP-linked anti-mouse antibody (
(Example 1-17) Application of Luminescence Probe Containing Luciferase (ALuc) of the Present Invention to ES Cell
(330) Novel ES cells expressing cSimgr13, which is an integrated-molecule-format bioluminescent probe containing the ALuc of Example 10 in its frame structure were established to visualize stress hormone sensitivity (
(331) One day after the subculturing of the transformed cells in a microslide, some of the cells in the wells were used as a control (0.1% DMSO), and the others were stimulated with 10.sup.5 M stress hormone (cortisol) for 20 minutes. Subsequently, after lysis, the cells were illuminated using a substrate-containing assay buffer in accordance with the protocol of luminescence assay kit produced by Promega, and the luminescence intensity was measured using an LAS-4000. Consequently, the group stimulated with stress hormone showed stronger luminescence than the control. The results indicated that the luminescence probe based on the luciferase (ALuc) of the present invention is also applicable to embryonic stem cells (ES), and does not lose hormone recognition ability.
(Example 1-18) Production of Additional Artificial Luciferases ALuc25 to ALuc32
(332) Additional functional artificial luciferases were developed based on the research and development results. Based on ALuc25, sequences having a large hydrophilic amino acid proportion and a small hydrophilic amino acid proportion were produced, and named ALuc26, ALuc27, ALuc28, and ALuc29. In addition, artificial luciferases having an antigen recognition site (epitope; examples including His tag, Myc tag, etc.) in the sequence were developed, and named ALuc30 (containing a His tag in the sequence), ALuc31 (containing an His tag in the sequence), and ALuc 32 (containing a Myc tag in the sequence) (
(333) After the insertion of a gene encoding an enzyme sequence as mentioned above into a mammalian expression vector (pcDNA3.1 (+)), each plasmid was introduced into COS-7 cells. By the transformation of these cells, each luciferase was expressed, and some of the luciferase was secreted out of the cells and some of the luciferase retained in the cells. 20 hours after the plasmid introduction, media of the cells were sampled, and the remaining cells were lysed with a lysis buffer (lysate preparation). Immediately after 50 L of assay solution (containing a substrate) was simultaneously added to each prepared medium (5 L) or lysate (5 L), the luminescence value was measured using a luminescence analyzer (LAS-4000; FujiFilm) (n=3) (
(334) Consequently, the luminescence values extremely higher than those of conventional GLuc and MpLuc4 were observed from the lysates of ALuc25, ALuc30, and ALuc31. In the cell medium group, strong bioluminescence was observed from the cell medium containing ALuc30 or ALuc31. The results indicated that ALuc30 and ALuc31 were better if strong bioluminescence from both conditions, i.e., the lysate and medium, were desired. In addition, since ALuc30 and ALuc31 contain an His tag in the sequence (
(335)
(336) Properties of the tag-including artificial luciferases after expression were evaluated using conventional Western blotting and affinity chromatography (
(337) First, each of the expression vectors of luciferases ALuc30 to 34 was introduced into COS-7 cells. One night after expression induction, the cell media were collected, and the presence of absence of secretion and the molecular weight of each expressed luciferase were respectively confirmed using affinity chromatography and Western blotting (
(338) The results found that His tag-containing ALuc30 was selectively extracted when each medium was purified using Ni-NTA affinity chromatography. The results of Western blotting using special antibodies (His tag antibody, HA tag antibody, Flag tag antibody) confirmed that the artificial luciferase (ALuc30, ALuc33, or ALuc34) was secreted in each medium and each tag functioned from the location of the band (indicating molecular weight) and the concentration of the band (indicating expression amount).
(Example 1-19) Prolonged Stability of Artificial Luciferase
(339) The prolonged activity stability of the artificial luciferases (ALucs) developed in the present invention was examined. First, a plasmid expressing each artificial luciferase (ALuc16, ALuc22, ALuc23, ALuc24, ALuc25, and ALuc30) (in the figure, each is referred to as A16, A22, A23, A24, A25, and A30) was introduced into COS-7 cells, and then culturing was continued for 24 hours. By the culturing, each artificial luciferase was secreted into the medium. The artificial luciferase secreted in the medium was collected, and a difference in enzymatic activity was measured under the same conditions in the following manner. On the first day (Day 0), 50 L of assay solution (containing native coelenterazine, produced by Promega) was added to 5 L of each medium, and the luminescence value (enzymatic activity) was measured using an LAS-4000 (produced by FujiFilm). In the same manner, the luminescence value on Day 8, Day 16, and Day 25 was compared with the luminescence value on Day 0 (
(340) For the medium samples 25 days after expression, temporal changes in luminescence intensity after substrate injection were observed in the following manner (
(341) The results indicated that the artificial luciferases of the present invention had excellent prolonged storage stability, because a remarkable inactivation phenomenon was not observed even after refrigerated storage for 25 days.
(Example 1-20) Living Cell Imaging Using ALuc of the Present Invention
(342) The following experiment was conducted to examine the living cell imaging abilities of the artificial luciferases established in the present invention (
(343) First, COS-7 cells were cultured in a microslide produced by ibidi. After the cells were raised to a certain level, the following genes were transferred to the cells in the channels. Channel group 1: pcDNA3.1 plasmid having a gene encoding a luminescent enzyme derived from Renilla reniformis (RLuc8.6-535); Channel group 2: pcDNA3.1 plasmid having a gene encoding A16-KDEL; and Channel group 3: pcDNA3.1 plasmid having a gene encoding a luminescent capsule based on ALuc16 (i.e., A16-MLS).
(344) After gene transfer, culturing was performed for another 16 hours. Immediately before imaging, the medium was substituted with an HBSS buffer containing a substrate, and the luminescence image obtained after the substitution was measured using an LAS-4000 (FujiFilm).
(345) Consequently, almost no luminescence image was obtained from the channels expressing RLuc8.6-535; however, a strong luminescence image was observed from the channels expressing A16-KDEL or A16-MLS (
(346) Similarly, COS-7 cells were cultured in a microslide produced by ibidi. After the cells were raised to a certain level, the following genes were transferred to the cells in the channels. Channel group 1: pcDNA3.1 plasmid having a gene encoding Cypridina luciferase; Channel group 2: pcDNA3.1 plasmid having a gene encoding A16-KDEL; and Channel group 3: pcDNA3.1 plasmid having a gene encoding a luminescent capsule based on A16 (i.e., A16-MLS).
(347) After gene transfer, culturing was performed for another 16 hours. 20 minutes before imaging, 40 L of a lysate buffer produced by Promega was added, and the resultant was allowed to stand for 20 minutes. Further, a substrate-containing assay buffer produced by Promega was added to the medium of each channel, and the luminescence image obtained after the addition was measured using an LAS-4000 (FujiFilm).
(348) Consequently, strong luminescence was only observed in channels of A16-KDEL and A16-MLS (
(Example 1-21) Establishment of Functional Artificial Luciferases (ALuc 30-34) Having Antigen Recognition Site (Epitope)
(349) Providing the artificial luciferase (ALuc) with functionality (antigen recognition ability, affinity chromatography purification ability) is an essential feature for the versatility of luminescent enzymes. To achieve this object, a series of artificial luciferases including a tag in part of the sequence of each artificial luciferase established in the present invention were established. These luciferases have a feature in that they contain an antigen recognition site (which can be also used for affinity chromatography purification) at an appropriate position in the sequence.
(350) CLUSTALW (http://www.genome.jp/tools/clustalw/), a known amino acid sequence alignment tool, was used in the example to determine the most suitable position at which to insert a tag. Specifically, the position of the tag was examined to exhibit 100% expected tag performance without inhibiting the luminescence activity of the enzyme (
(351) To compare the luminescence intensity of novel luciferases, a comparison experiment was performed under the same conditions as Example 1-2 (COS-7 cells, culturing for one day, using an assay kit produced by Promega) (
(Example 1-22) Stability of Functional Artificial Luciferase Having Antigen Recognition Site (Epitope)
(352) The luminescence characteristics of the novel functional artificial luciferases (containing an antibody recognition site (epitope sequence), etc.) established in Example 1-21 were examined.
(353) To compare the relative luminescence intensity of these luciferases, the following experiment was conducted. First, a gene encoding each luciferase was subcloned into pcDNA3.1 (+), which is a eukaryotic expression vector, and each vector was introduced into COS-7 cells. Two days after gene transfer, the relative luminescence intensity of luciferase secreted into each medium was compared using a luminescence reagent produced by Promega.
(354) The results surprisingly confirmed a phenomenon in which the luminescence intensity of some luciferases was gradually increased after the substrate injection (
(355) The results of the experiment indicated that the luciferases containing the antibody recognition site obtained herein can, by the antibody recognition site, function as luminescent labels in a living body without losing their enzymatic activity.
(Example 2-1) to (Example 2-10)
(356) The following shows examples of the chemical structures of the components used in (Example 2-1) to (Example 2-10) described below.
(357) ##STR00001##
(Example 2-1) Comparative Study on Combination of Lysis Buffer and Assay Buffer
(358) In this example, a comparative study of luminescence intensity was made on different combinations of a lysis buffer and an assay buffer used in conventional bioassays (
(359) The results revealed that the systems for which the luciferase lysis buffer (cat. E291A) from Promega and a C3 cell lysis solution were used showed outstandingly high luminescence intensities. The results also indicated that HESS and TE PEG buffers are useful as an assay buffer. In particular, when an HESS buffer or a TE PEG buffer was used as an assay buffer with the cell lysis solution of Promega (cat. E291A), high luminescence stability was observed (89 to 92%) (
(Example 2-2) Search for Optimal Assay Buffer Compatible with C3
(360) On the basis of the results of Example 2-1 indicating C3 as an effectual cell lysis buffer, a search for an optimal assay buffer compatible with C3 was further conducted (
(361) As seen in
(Example 2-3) Analysis of Effects of Heavy Metal Ions as Additive for Assay Buffer
(362) The results of Examples 2-1 and 2-2 revealed that C3 is a useful cell lysis buffer, and that PBS and HBSS are useful assay buffers.
(363) This example examined the effects of a heavy metal ion as an additive for an assay buffer (
(364) As seen from
(Example 2-4) Effect of Adding Glycols to Bioassay Reaction Solution
(365) This example examined the effects of adding a glycol to a bioassay reaction solution by actually using a luminescent probe (
(366) In accordance with a procedure for preparing an integrated-molecule-format bioluminescent probe developed by the present inventors (Non-patent Document 28), a novel integrated-molecule-format bioluminescent probe was developed. An artificial luciferase (ALuc) (for which a patent application was filed on the same date as the application date of the present invention) was newly developed by the present inventors following a technique in which many plankton-derived luciferases were bundled, and from the bundle, thermodynamically highly stable sequences were extracted. The artificial luciferase (ALuc) was bisected, and then an androgen receptor (AR LED) was inserted therebetween (
(367) For this example, first, COS-7 cells were cultured in a 96-well plate, and a plasmid for encoding the integrated-molecule-format bioluminescent probe was added thereto. After 16-hour incubation in a CO.sub.2 incubator, 50 L of a C3 solution was added to each well to allow for cell lysis for 20 minutes. HESS buffers each containing a substrate and PPG or PEG in the amounts shown in
(368) The results revealed that the addition of PPG or PEG is expected to produce effective results. It was also found that the suitable amount of such additives is about 1% based on the amount of the assay buffer.
(Example 2-5) Effect of Adding Halogen Ions to Bioassay Reaction Solution
(369) This example examined the effects of adding a halogen ion to a bioassay reaction solution (
(370) A plasmid for encoding the integrated-molecule-format bioluminescent probe was introduced into COS-7 cells cultured in a 96-well plate using TranslT-LT1. After 16-hour incubation in a CO.sub.2 incubator, 50 L of a C3 solution was added to each well to allow for cell lysis for 20 minutes. HESS buffers containing a substrate and KBr or KI in the amounts shown in
(371) The results revealed that the addition of a halogen ion is effective. Specifically, KI in any amount achieved excellent results, and the addition of KI in an amount of 50 mM achieved particularly excellent results. KBr, when added in an amount of about 100 mM, achieved excellent results.
(Example 2-6) Effect of Adding Polysaccharides to Bioassay Reaction Solution
(372) This example examined the effects of adding a polysaccharide to a bioassay reaction solution (
(373) A plasmid for encoding the integrated-molecule-format bioluminescent probe was introduced into COS-7 cells cultured in a 96-well plate using TranslT-LT1. After 16-hour incubation in a CO.sub.2 incubator, 50 L of a C3 solution was added to each well to allow for cell lysis for 20 minutes. HESS buffers containing a substrate and polysaccharides in the amounts shown in the figure were prepared, and 50 L of each of the buffers was added to the lysate simultaneously with a multichannel pipette. The luminescence was immediately measured using a conventional luminometer (GloMax 20/20n; Promega).
(374) The results revealed that the addition of sucrose and glucose is particularly effective. The addition of not only a polysaccharide, but also glycine was found to be effective. The suitable amount was 2 mg/mL.
(Example 2-7) Stress Hormone Assay Test 1 Using Integrated-Molecule-Format Bioluminescent Probe (cSimgr8)
(375) To demonstrate the excellent performance of the one-shot buffer according to the present invention, bioassays were conducted by using C14 to C22 reaction buffers of the present invention with an integrated-molecule-format bioluminescent probe (cSimgr8) having a stress hormone receptor (the ligand-binding domain of glucocorticoid receptor; GR LBD) as a frame structure (
(376) First, COS-7 cells were cultured in a 96-well plate, and cSimgr8 was introduced thereto. After an additional 16-hour culture, the COS-7 cells in the plate were divided into two groups. One group of the cells was stimulated with 10.sup.5 M of a stress hormone (cortisol) for 20 minutes, and the other group of the cells was simulated with a control (0.1% DMSO) for 20 minutes. Thereafter, the medium in the plate was discarded to leave only the cells in the plate. Buffers of the formulations of C14 to C22 were prepared as one-shot buffers. A high-sensitivity Promega luminometer (GloMax 20/20n; Promega) was used for luminescence measurement.
(377) The assay was conducted as follows. C14 to C22 solutions (50 L each) were individually added to the wells of the plate from which the medium was already removed, and the samples were pipetted a few times. Thereafter, the whole volume of the samples was individually placed into respective 1.6 mL Eppendorf tubes, and the amount of light was measured for 3 seconds with the luminometer.
(Example 2-8) Stress Hormone Assay Test 2 Using Integrated-Molecule-Format Bioluminescent Probe (cSimgr8)
(378) A stress hormone assay using an integrated-molecule-format bioluminescent probe (cSimgr8) was conducted in the same manner as in Example 2-7 using C23 to C26 reaction buffers of the present invention (
(379) As in Example 2-7, COS-7 cells were cultured in a 96-well plate, and cSimgr8 was introduced thereto. After an additional 16 hour-culture, the COS-7 cells in the plate were divided into two groups. One group of the cells was stimulated with 10.sup.5 M of a stress hormone (cortisol) for 20 minutes, and the other group of the cells was simulated with a control (0.1% DMSO) for 20 minutes. Thereafter, the medium in the plate was discarded to leave only the cells in the plate. Buffers of the formulations of C14 to C22 were prepared as one-shot buffers. A high-sensitivity Promega luminometer (GloMax 20/20n; Promega) was used for luminescence measurement.
(380) The assay was conducted as follows. C23 to C26 solutions (50 L each) were individually added to the wells of the plate from which the medium was already removed, and the samples were pipetted a few times. Thereafter, the whole volume of the samples was individually placed into respective 1.6 mL Eppendorf tubes, and the amount of light was measured for 3 seconds with the luminometer.
(Example 2-9) Androgen Assay Test Using Integrated-Molecule-Format Bioluminescent Probe (pLeu-rich)
(381) To demonstrate the excellent performance of the one-shot buffer according to the present invention, the buffer was used for an integrated-molecule-format bioluminescent probe (pLeu-rich) having an androgen (male hormone) receptor (the ligand-binding domain of androgen receptor; AR LBD) as a frame structure. pLeu-rich is an integrated-molecule-format bioluminescent probe that was previously developed by the present inventors using a firefly-derived luciferase (Non-patent Document 29).
(382) COS-7 cells were cultured in a 96-well plate, and pLeu-rich was introduced thereto. After an additional 16-hour culture, the COS-7 cells in the plate were divided into two groups. Some portions of the cells were individually stimulated with 10.sup.5 M of 5-dihydrotestosterone (DHT), 4-hydroxytamoxifen (OHT), 17-estradiol (E.sub.2), dichlorodiphenyltrichloroethane (DDT), or polychlorinated biphenyls (PCB) for 20 minutes. Thereafter, the medium was discarded, and the assay preparation for the cells was completed. A reaction buffer prepared by mixing a C29 solution with D-luciferin was used. A high-sensitivity Promega luminometer (GloMax 20/20n; Promega) was used for luminescence measurement.
(383) The assay was conducted as follows. 50 L of a C16 solution was added to the plate from which the medium was already removed, and the samples were pipetted a few times. Thereafter, the whole volume of the samples were individually placed into respective 1.6 mL Eppendorf tubes, and the amount of light was measured for 3 seconds with the luminometer.
(384)
(Example 2-10) Estrogen Assay Test Using Integrated-Molecule-Format Bioluminescent Probe (pSimer-r2)
(385) To demonstrate the excellent performance of the reaction buffer (one-shot buffer), the buffer was used for an integrated-molecule-format bioluminescent probe (pSimer-r2) having an estrogen (female hormone) receptor (the ligand-binding domain of estrogen receptor; ER LBD) as a frame structure. pSimer-r2 is an integrated-molecule-format bioluminescent probe that was previously developed by the present inventors using a luciferase originating from click beetle (click beetle luciferase; CBLuc) (Non-patent Document 12).
(386) COS-7 cells were cultured in a 96-well plate, and pSimer-r2 was introduced thereto. After an additional 16-hour culture, the COS-7 cells in the plate were divided into two groups. Some portions of the cells were individually stimulated with 10.sup.5 M of 5-dihydrotestosterone (DHT), 4-hydroxytamoxifen (OHT), 17-estradiol (E.sub.2), dichlorodiphenyltrichloroethane (DDT), or polychlorinated biphenyls (PCB) for 20 minutes. The remaining cells were used as a control group (control). Thereafter, the medium was discarded, and the assay preparation for the cells was completed. C16 was used as a one-shot reaction buffer. A high-sensitivity Promega luminometer (GloMax 20/20n; Promega) was used for luminescence measurement.
(387) The assay was conducted as follows. 50 L of a C16 solution was added to the plate from which the medium was already removed, and the samples were pipetted a few times. Thereafter, the whole volume of the samples were individually placed into respective 1.6 mL Eppendorf tubes, and the amount of light was measured with the luminometer.
(388)
INDUSTRIAL APPLICABILITY
(389) By using the present invention for ligand measurement based on a reporter-gene assay method, which has hitherto been widely used, or as a replacement for a luciferase used in a known bioluminescent probe, it becomes possible to exponentially improve measurement performance during assay. Therefore, the present invention can be used for various applications, including the development of a diagnosis reagent for basic biology research, medical and pharmaceutical purposes, or analytical chemistry.
(390) Sequence Listing