Luminescent substrate for use in artificial bioluminescent enzyme
10214766 ยท 2019-02-26
Assignee
Inventors
- Sung Bae Kim (Tsukuba, JP)
- Hiroshi Izumi (Tsukuba, JP)
- Hiroaki Tao (Tsukuba, JP)
- Masaki Torimura (Tsukuba, JP)
- Akihiro Wakisaka (Tsukuba, JP)
Cpc classification
C12Y113/12007
CHEMISTRY; METALLURGY
C12Y113/12
CHEMISTRY; METALLURGY
C12N9/0069
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a bioluminescent substrate suitably usable in a series of artificial luciferases (ALuc), and the invention provides a wavelength-shifted spectrum with a selective high intensity luminescence and high luminescence stability obtained by the use of the substrate together with ALuc. The luminescent substrate for ALuc obtained by the invention can be included together with a suitable luminescence solution in a luminescence kit. The bioluminescent substrate for ALuc of the invention can exhibit unprecedented excellent luminescence specificity and functionality in the conventional bioluminescence probe, two-hybrid assay, bioluminescent capsule, and reporter gene assay.
Claims
1. A method for detecting luminescence comprising (a) contacting a compound of formula (1) with polypeptide (B), and (b) measuring the intensity of luminescence generated by the contact, wherein formula (1) is: ##STR00009## wherein X.sup.a represents a phenyl group optionally substituted with halogen or a naphthyl group, X.sup.b represents a phenyl group, and M.sup.c represents hydrogen, hydroxy, or thiol, and wherein polypeptide (B) has a copepod luciferase activity and is chosen from the group consisting of: (i) the amino acid sequence of SEQ ID No: 1; and (ii) the amino acid sequence of SEQ ID No: 1 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.
2. A kit for measuring bioluminescence comprising (a) a compound represented by formula (1) ##STR00010## wherein X.sup.a represents a phenyl group optionally substituted with halogen or a naphthyl group, X.sup.b represents a phenyl group, and M.sup.c represents hydrogen, hydroxy, or thiol and (b) a polypeptide having a copepod luciferase activity chosen from the group consisting of: (i) the amino acid sequence of SEQ ID No:1, and (ii) the amino acid sequence of SEQ ID No: 1 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.
3. A method comprising the method according to claim 1, further in combination with one or more additional bioluminescent enzymes.
4. The method according to claim 1, which is a reporter gene assay, a two-hybrid assay, a bioluminescent capsule assay, or an integrated-molecule-format bioluminescent probe measurement method.
5. A bioluminescence resonance energy transfer (BRET) method comprising (a) contacting a compound represented by formula (1) with polypeptide (B), (b) allowing bioluminescent energy generated by the contact to transfer to another fluorescence protein, and (c) measuring the luminescence intensity of the fluorescence protein to which the bioluminescent energy has transferred, wherein formula (1) is represented by: ##STR00011## wherein X.sup.a represents a phenyl group optionally substituted with halogen or a naphthyl group, X.sup.b represents a phenyl group, and M.sup.c represents hydrogen, hydroxy, or thiol, and wherein polypeptide (B) has a copepod luciferase activity and is chosen from the group consisting of: (i) the amino acid sequence of SEQ ID No: 1; and (ii) the amino acid sequence of SEQ ID No: 1 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.
6. The method according to claim 1, wherein the compound represented by formula (1) is coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), or coelenterazine 400A (CTZ 400A).
7. The kit according to claim 2, wherein the compound represented by formula (1) is coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), or coelenterazine 400A (CTZ 400A).
8. The method according to claim 3, which is a reporter gene assay, a two-hybrid assay, a bioluminescent capsule assay, or an integrated-molecule-format bioluminescent probe measurement method.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DESCRIPTION OF EMBODIMENTS
(11) [I] Artificial bioLuminescent Enzyme and Luminescent Substrate
(12) 1. Optimum Luminescent Substrate for Artificial bioLuminescent Enzymes (ALuc)
(13) Luminescent Substrate
(14) The luminescent substrate of the present invention is a compound represented by the following formula (1).
(15) ##STR00007##
wherein X.sup.a represents a phenyl group optionally substituted with one halogen atom or naphthyl group (naphthalene). The position of the substituent (ortho-, meta-, or para-) is not limited. However, when the phenyl group has a substituent, the position is preferably meta- or para-, and particularly preferably para-.
(16) As used herein, the halogen atom refers to chlorine, bromine, fluorine, or iodine.
(17) X.sup.b represents a phenyl group.
(18) M.sup.c represents one hydrogen atom, hydroxyl group, or thiol group. The position of hydroxy or thiol groups on the benzene ring (ortho-, meta-, or para-) is not limited, but is preferably meta- or para-, and particularly preferably para-.
(19) Examples of the compound of the present invention include typically coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), and coelenterazine 400A (CTZ 400A). Although CTZ n, CTZ i, CTZ f, and CTZ h are particularly preferable, the compound is not limited thereto.
(20) The following shows the chemical structural formulae of coelenterazine n (CTZ n), coelenterazine i (CTZ i), coelenterazine f (CTZ f), coelenterazine h (CTZ h), and coelenterazine 400A (CTZ 400A).
(21) ##STR00008##
(22) The luminescent substrate of the present invention can be synthesized by, for example, following a synthesis route disclosed in Non-patent Literature 17, 18, 24, 26, 28, or 29.
(23) The luminescent substrate can also be synthesized by cross coupling (Stille coupling) catalyzed by palladium as disclosed in Non-patent Literature 32, 33, or 34.
(24) Artificial bioLuminescent Enzyme
(25) In the present invention, the luminescent substrates are used for the following artificial bioluminescent enzymes (which may be referred to sometimes as artificial luciferase or ALuc).
(26) Examples of typical artificial luciferases (ALuc) of the present invention include ALuc10 (SEQ ID NO: 4), ALuc15 (SEQ ID NO: 5), ALuc16 (SEQ ID NO: 6), ALuc17 (SEQ ID NO: 7), ALuc18 (SEQ ID NO: 8), ALuc19 (SEQ ID NO: 9), ALuc21 (SEQ ID NO: 10), ALuc22 (SEQ ID NO: 11), ALuc23 (SEQ ID NO: 12), Luc24 (SEQ ID NO: 13), ALuc25 (SEQ ID NO: 14), ALuc26 (SEQ ID NO: 15), ALuc27 (SEQ ID NO: 16), ALuc28 (SEQ ID NO: 17), ALuc29 (SEQ ID NO: 18), ALuc30 (SEQ ID NO: 19), ALuc31 (SEQ ID NO: 20), ALuc32 (SEQ ID NO: 21), ALuc33 (SEQ ID NO: 22), and ALuc34 (SEQ ID NO: 23).
(27) The artificial luciferase (ALuc) of the present invention can be expressed as a polypeptide comprising an amino acid sequence of any one of Items (i) to (iii) below and having copepod luciferase activity:
(28) (i) an amino acid sequence represented by any of SEQ ID NOs: 4 to 23;
(29) (ii) an amino acid sequence represented by any of SEQ ID NOs: 4 to 23 in which one or several amino acids are deleted, substituted, inserted, or added,
in which several means 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acids);
(iii) an amino acid sequence having an identity of not less than 90% with any of amino acid sequences represented by SEQ ID NOs: 4 to 23.
(30) 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.
(31) The amino acid sequences of the artificial luciferases (ALucs) of the present invention have common basic frame structures shown in
(32) (iv) the amino acid sequence represented by SEQ ID NO: 2;
(33) (v) an amino acid sequence represented by SEQ ID NO: 2 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;
(34) (iv) the amino acid sequence represented by SEQ ID NO: 1;
(35) (v) an amino acid sequence represented by SEQ ID NO: 1 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;
(36) (vi) the amino acid sequence represented by SEQ ID NO: 3; or
(37) (vii) an amino acid sequence represented by SEQ ID NO: 3 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.
(38) In the amino acid sequence represented by SEQ ID NO: 3, 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: 2, 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: 1. 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 excluded.
(39) The function of artificial bioluminescent enzyme is not significantly impaired even when the region of positions 20 to 29 in the amino acid sequence represented by SEQ ID NO: 3 (corresponding to a region of positions 20-29 in the amino acid sequence represented by SEQ ID No. 2, and a region of positions 20-31 in the amino acid sequence represented by SEQ ID NO: 1) is substituted with a functional amino acid sequence (e.g., antigen recognition site, affinity chromatography recognition site, or localization signal). Accordingly, part or all of the amino acids in this region may be deleted.
(40) In the amino acid sequences represented by SEQ ID NOs: 1 to 3, amino acids represented by Xaa are explained in detail below.
(41) Of the amino acids represented by Xaa in SEQ ID NO: 2, 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 is E or G; positions 20-27 are PTENKDDI (SEQ ID NO: 26), ATINEEDI (SEQ ID NO: 27), ATINENFE (SEQ ID NO: 28), HHHHHHHH (SEQ ID NO: 29), EKLISEE (SEQ ID NO: 30), MMYPYDVP (SEQ ID NO: 31), or MMDYKDDD (SEQ ID NO: 32); 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: 33), IVGA (deletion of four residues, SEQ ID NO: 34), ITEEE (deletion of three residues, SEQ ID NO: 35), or IGGPIVD (SEQ ID NO: 36); 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.
(42) 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.
(43) 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.
(44) 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.
(45) 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.
(46) 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.
(47) Of the amino acids represented by Xaa in SEQ ID No. 1, 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: 37), ATINEEDI (deletion of two residues, SEQ ID NO: 38), ATINENFEDI (SEQ ID NO: 39), HHHHHHHH (deletion of two residues, SEQ ID NO: 40), EKLISEE (deletion of two residues, SEQ ID NO: 41), MMYPYDVP (deletion of two residues, SEQ ID NO: 42), or MMDYKDDD (deletion of two residues, SEQ ID NO: 43); 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 KI; position 129 is E, G, or A; positions 140-144 are TEEET (SEQ ID NO: 44), GEAI (deletion of one residue, SEQ ID No. 45), or VGAI (deletion of one residue, SEQ ID NO: 46); positions 148-151 are GVLG (SEQ ID NO: 47) 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.
(48) 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.
(49) 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.
(50) 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.
(51) 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.
(52) 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.
(53) Of the amino acids represented by Xaa in SEQ ID NO: 3, 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.
(54) 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.
(55) Amino acids at positions 5, 24, 25, 60, 64, 65, 70, 95, 108, 153, 200, and 208 are hydrophilic 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.
(56) Typical examples of the amino acid sequence represented by SEQ ID NO: 3 include ALuc10, ALuc15, ALuc16, ALuc18, ALuc22, ALuc23, and ALuc25.
(57) One embodiment of the artificial luciferase of the present invention includes the amino acid sequence represented by SEQ ID NO: 24 as the region corresponding to positions 1-71 in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to the region of positions 1-69 in the amino acid sequence represented by SEQ ID NO: 2, and the region of positions 1-69 in the amino acid sequence represented by SEQ ID NO: 3). Typical examples include ALuc15, ALuc16, ALuc17, ALuc18, and ALuc24.
(58) Another embodiment of the artificial luciferase of the present invention includes the amino acid sequence represented by SEQ ID NO: 25 as the region corresponding to positions 1-157 in the amino acid sequence represented by SEQ ID NO: 1 (corresponding to the region of positions 1-155 in the amino acid sequence represented by SEQ ID NO: 2, and the region of positions 1-152 in the amino acid sequence represented by SEQ ID NO: 3). Typical examples include ALuc22, ALuc25, ALuc26, ALuc27, ALuc28, and ALuc29.
(59) 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.
(60) Specifically, in the artificial luciferase embedding an antibody recognition site (epitope sequence) therein, a region corresponding to positions 20-29 in SEQ ID NO: 2 or a region corresponding to positions 20-31 in SEQ ID NO: 1 includes an antibody recognition site (epitope sequence). Preferable examples of the antibody recognition site (epitope sequence) include His-tag (HHHHHH) (SEQ ID NO: 48), FLAG-tag (DYKDDDDK) (SEQ ID NO: 49), Myc-tag (EQKLISEEDL) (SEQ ID NO: 50), and HA-tag (YPYDVPDYA) (SEQ ID NO: 51); however, the antibody recognition site is not limited thereto.
(61) In an example of the artificial luciferase embedding a His-tag therein, amino acids at positions 20-29 in SEQ ID NO: 2 or amino acids at positions 20-31 in SEQ ID NO: 1 are all H (His8 sequence). Typical examples include ALuc30 and ALuc31.
(62) In an example of the artificial luciferase embedding a c-Myc-tag therein, the sequence of the region corresponding to positions 20-29 in SEQ ID NO: 2 or the sequence of the region corresponding to positions 20 to 31 in SEQ ID NO: 1 is EQKLISEEDL (Myc-tag sequence, SEQ ID NO: 50). Typical examples include ALuc32.
(63) In an example of the artificial luciferase embedding an HA-tag therein, amino acids at positions 20-29 in SEQ ID NO: 2 or amino acids at positions 20-31 in SEQ ID NO: 1 are YPYDVPDYA (HA-tag sequence, SEQ ID NO: 51). Typical examples include ALuc33.
(64) In an example of the artificial luciferase embedding a FLAG-tag therein, amino acids at positions 20-29 in SEQ ID NO: 2 or amino acids at positions 20-31 in SEQ ID NO: 1 are DYKDDDDK (FLAG-tag sequence, SEQ ID NO: 49). Typical examples include ALuc34.
(65) As used herein, copepod luciferase refers to a bioluminescent enzyme (luciferase) produced by tiny Crustaceans, luminescent plankton called copepods. Specific examples include MoLuc1, MoLuc2, PaLuc1, PaLuc2, LoLuc, HtLuc1, HtLuc2, HmLuc1, HmLuc2, Gaussia luciferases (GLuc), and copepod luciferases (MLuc, MpLuc1, MpLuc2). The substrate specificity of copepod luciferases is that the luciferases specifically oxidize coelenterazine. Typically, copepod luciferases catalyze a luminescent reaction in the deep-sea environment, specifically, at an optimum pH of about 7.5 to 8 and at an optimum temperature of about 4 to 10 C. (enzymatic properties), but also extensively catalyze luminescence reactions under different conditions. As used herein, copepod luciferase refers to a luciferase having an enzyme activity and structure in common with luciferases derived from known copepods. Specifically, the copepod luciferase in the present specification has enzyme activity for catalyzing a luminescent reaction at an optimum pH of about 5 to 8 and at an optimum temperature of about 4 to 25 C. in the presence of coelenterazine serving as a substrate, and the luciferase has two enzyme activity domains and a secretion signal at N-terminus, with a molecular weight of about 20 kD (18 kD-28 kD), which is the smallest among bioluminescent enzymes.
(66) The coelenterazine is not limited to native coelenterazine (native CTZ, n CTZ), and also encompasses a variety of derivatives of native coelenterazine. Specifically, coelenterazine can also be referred to as coelenterazines. 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), and coelenterazine n (CTZ n).
(67) 2. Luminescent Performance Evaluation on Optimum Luminescent Substrate of the Present Invention Used for Artificial Luciferase (ALuc)
(68) (2-1) Enzyme Activity Confirmation Method
(69) The enzyme activity of ALuc in the presence of an optimum luminescent substrate can be examined, for example, according to the following method.
(70) First, using a known lipid reagent for gene introduction, a eukaryotic cell expression vector (e.g., pcDNA3.1(+)) encoding ALuc is introduced into African monkey-derived COS-7 cells; as a control, an expression vector encoding a known copepod bioluminescent enzyme 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, the cells are individually dissolved in a known cell lysis solution.
(71) An example of base sequences encoding ALuc is shown by the sequence number.
(72) Thereafter, the cell lysis solution is mixed with a known buffer solution containing the optimum luminescent substrate of the present invention, and its color intensity, temporal stability in luminescence, etc., are measured.
(73) The luminescence intensity may be determined by measuring the intensity at a specific wavelength using a known luminescence spectrophotometer after the addition of the optimum luminescent substrate of the present invention. Measuring the luminescence intensity every minute shows the temporal change in luminescence to thereby enable the evaluation of luminescence stability. To measure a shift to red light, the entire wavelength is scanned.
(74) As described above, the substrate of the present invention exhibited 100 to 10,000 times higher selectivity to artificial luciferases (ALuc) developed by the present inventors than to known marine animal-derived luciferase. This indicates that the use of the optimum luminescent substrate of the present invention provides assurance of luminescence specificity in various bioassay systems using ALuc.
(75) Hereinafter, a reporter analysis method in which the optimum luminescent substrate for ALuc is usable in the present invention is categorized into three groups: basic, inducible, and activatable, which are disclosed in Non-patent Literature 16 by Niu et al. 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 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 integrated-molecule-format bioluminescent probe and bioluminescent capsule. This method can also be applied to protein complementation assay (PCA), protein splicing assay (PSA), etc.
(76) (2-2) Basic Method
(77) When the ALuc of the present invention is applied to a basic method as a reporter protein, a fusion protein may be prepared by simply linking the ALuc to a target protein. The basic method differs from other reporter analysis methods in that expression during the preparation of the fusion protein is performed by using an uncontrollable promoter.
(78) 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.
(79) 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.
(80) (2-3) Inducible Method
(81) 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 ALuc as a reporter gene essential for these analysis systems remarkably improves assay measurement performance.
(82) Hereinafter, the reporter gene assay and the two-hybrid assay, which are typical inducible method analysis systems, are explained in detail.
(83) (i) Reporter Gene Assay
(84) 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 intensity of bioluminescence.
(85) 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.
(86) In the reporter gene assay, firefly luciferase that has been widely used has the following drawbacks: (i) due to its large molecular weight, the maturation of expressed mRNA 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 ALuc as a reporter protein, these problems are overcome.
(87) Since the use of the luminescent substrate of the present invention with ALuc as a reporter protein ensures a significantly high luminescence intensity of the reporter, it has an advantage of very early stage 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 allows its improved transmittance through the plasma membrane or skin, the background intensity level is reduced, and high measurement accuracy can be achieved.
(88) More specifically, ALuc, together with the luminescent substrate of the present invention, is employed in these reporter gene assays in such a manner that the bioluminescent 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 of bioluminescence 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.
(89) (ii) Two-Hybrid Method
(90) 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.
(91) 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.
(92) Specifically, examples of the vector that has a DNA encoding a DNA binding region of transcriptional regulator GAL4 and that can use 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 pVP16 (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.
(93) In this case, for example, a vector in which ALuc gene 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), ALuc, together with the substrate 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).
(94) (2-4) Activatable Method
(95) 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 ALuc 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 (bioluminescent capsule) previously developed is explained. In addition, ALuc 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. These methods may sometimes be referred to as bioluminescent capsule method or bioluminescent capsule assay in the present specification.
(96) (i) Production of Luminescent Fusion Protein (Bioluminescent Capsule)
(97) By binding a membrane localization signal (MLS) to the C-terminus of ALuc, the ALuc can be localized in the plasma membrane. 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 sequence actively 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 bioluminescent capsule.
(98) Compared to conventional bioluminescent probes, the bioluminescent capsule shows stable optical properties with remarkably high intensity, and is responsive to a specimen that cannot pass through the plasma membrane. The bioluminescent capsule has a structure in which a membrane localization signal (MLS) is linked to the C-terminus of the bioluminescent enzyme as a basic frame structure. Since the effect of a chemical causing a conformational change on the cell surface, such as a chemical inducing cell death, can be visualized as a conformational change in the plasma membrane surface, by this structure or even when ALuc is linked to a tandem to enhance the intensity of luminescence, easy observation is possible. Preferably, it is possible to insert between the MLS and the C-terminus of the bioluminescent enzyme, a polypeptide causing a conformational 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 bioluminescent 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 ISTD used in Examples 1-7 is inserted as a cargo, cell death can be visualized by caspase-3 activities. Further, by linking a fluorescence protein or another bioluminescent enzyme as a cargo between the bioluminescent enzyme and MLS, the intensity of luminescence on the plasma membrane surface is increased as in the case where the bioluminescent enzyme together with the luminescent substrate of the present invention is linked in 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.
(99) The bioluminescent capsule 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 ALuc. Typical examples include
(100) (a) a luminescent fusion protein wherein a fluorescence protein or luciferase is inserted between the membrane localization signal (MLS) and the C-terminus of ALuc (the luciferase may be other ALuc), and
(101) (b) a luminescent fusion protein wherein a polypeptide changing the conformation in the plasma membrane, or a polypeptide having or less amino acids, preferably 10 or less amino acids recognized by the polypeptide changing the conformation in the plasma membrane, is inserted between the membrane localization signal (MLS) and the C-terminus of ALuc. The polypeptide changing the conformation in the plasma membrane is particularly preferably a polypeptide inducing cell death, and more preferably a polypeptide having 20 or less amino acids containing the recognition sequence of caspases, i.e., DEVD or ISTD.
(ii) Application to Bioluminescent Probe
(102) Further, by incorporating ALuc into the integrated-molecule-format bioluminescent 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 bioluminescent probe (Non-patent Literature 7 and Non-patent Literature 8), which are recited in the pending patents applied by the present inventors, the presence or absence of a ligand and the intensity of the ligand activity can be observed with high luminance. By comprising, as the probe components, (i) the bisected bioluminescent 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 interaction of the ligand with the ligand-binding protein, which are linked to the vicinity of the bisected bioluminescent enzyme, it is possible to form a high-performance bioluminescent probe. This bioluminescent 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 enzyme 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.
(103) In the present invention, integrated molecule-format bioluminescent 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 bioluminescent probe denotes a fusion protein that comprises, as fundamental components, the two fragments of N- and C-terminals obtained by bisecting ALuc, a ligand-binding protein, and a recognition protein for recognizing the ligand-binding protein. Similarly, two molecule-format bioluminescent probe in the present invention denotes a bioluminescent probe in which the two fragments of N- and C-termini obtained by bisecting ALuc are present in the fusion protein containing the ligand-binding protein, and in the fusion protein containing the recognition protein, respectively.
(104) When ALuc is used for these bioluminescent probes, the ALuc must be bisected into an N-terminal fragment and a C-terminal fragment.
(105) Patent Literature 1 to 4 discloses the details regarding the actual method for using ALuc as an integrated molecule-format bioluminescent probe. More specifically, ALuc is bisected, and a chimera DNA encoding a bioluminescent 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 tandemly linked. 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 mammal-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.).
(106) Since the bioluminescent probe using the superluminescent enzyme together with the optimum luminescent substrate according to the present invention is introduced into cells as a chimera DNA and expressed in the cells as a fusion protein, by measuring the variance in light intensity emitted from the cells after subjecting the transformed cell to ligand stimulation, the property or levels of activity of the ligand may be evaluated.
(107) When ALuc 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 interaction with the ligand, for example, change the steric conformation, 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 an extracellular stimulus, or an intracellular ligand that is produced inside the cells in response to the extracellular stimulus. Examples thereof include agonists or antagonists of the receptor protein (for example, nuclear receptor, or G-protein-coupled receptor), signal transduction proteins such as cytokine, chemokine, or insulin, intracellular second messenger, lipid second messenger, phosphorylated amino acid residue, G-protein-coupled receptor ligand, and like ligands that specifically bind to proteins involved in intracellular signal transduction.
(108) 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 the intracellular signal transduction substance that is newly produced as a result of the interaction 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.
(109) (iii) Intracellular Imaging
(110) 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 cell components 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.
(111) This process is performed according to the method of Yamanaka et al. (Non-patent Literature 36).
(112) Further, by linking the ALuc to a suitable signal peptide, the ALuc can be used for high luminance imaging of various organelles. For example, by linking a GAP-43-derived MLCCMRRTKQV sequence (SEQ ID NO: 52) to the N- or C-terminus of ALuc, the ALuc may be localized in the plasma membrane. Linking a GRKKRRQRRR sequence (SEQ ID NO: 53) to a terminus enables localization in the cytosolic compartment of cells. Further, for localization in the endoplasmic reticulum (ER) and the cellular nucleus, KDEL (SEQ ID NO: 54) and DPKKKRKV (SEQ ID NO: 55) sequences, respectively, are linked to a terminus. Furthermore, by linking to HIS-tag (HHHHHH) (SEQ ID NO: 48), FLAG-tag (DYKDDDDK) (SEQ ID NO: 49), Myc-tag (EQKLISEEDL) (SEQ ID NO: 50), HA-tag (YPYDVPDYA) (SEQ ID NO: 51), V5-tag (GKPIPNPLLGLDST) (SEQ ID NO: 556), T7-tag (MASMTGGQQMG) (SEQ ID NO: 57) 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.
(113) [II] Determination of Reaction Solution Containing Luminescent Substrate of the Present Invention Used for Bioassay
(114) 1. Reaction Solution for Bioassay
(115) (1-1) Lysis Buffer (Cell Lysis Solution) and Assay Buffer (Reaction Solution)
(116) Conventionally conducted bioassays involve two separate assay buffers: a buffer for lysis (cell lysis solution); and a buffer for assay (assay solution). This is because high lytic activity and low inhibitory effect on a bioluminescent 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.
(117) 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.
(118) The following description describes a study on the formulations of the reaction solution components usable together with the optimum luminescent substrate for ALuc in the present invention.
(119) (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-(357 Da), cystatin, bestatin, epibestatin hydrochloride, aprotinin, minocycline, ALLN (384 Da), and the like (h) 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-ethylmorpho line, 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
(i) Others: sodium molybdate (stabilization of receptors), dithiothreitol (dithiothreitol, DTT) (reducing agent)
(1-2) Basic Reaction Solution Component 1 to which the Optimum Luminescent Substrate for ALuc of the Present Invention can be Added
(120) 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.
(121) First, the following four types of solutions are prepared beforehand, and mixed for use. Solution 1: 1.4% NaHCO.sub.3 solution 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 Solution 3: a solution prepared by dissolving 1.4 g of CaCl.sub.2 in 100 ml of water 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
(122) The mixture is adjusted to a pH of 7.0 with a sodium hydroxide solution (N/20) so as to give 200 ml.
(123) 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.
(124) (1-3) Basic Reaction Solution Component 2 to which the Optimum Luminescent Substrate for ALuc of the Present Invention can be Added
(125) 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.
(126) As the reaction solution in the present invention, a Tris buffer can be suitably used for bioluminescence, and can be a basic buffer component usable in both lysis and assay.
(127) (1-4) Buffer Formulation in the Present Invention
(128) The above-stated basic buffer components, an HBSS 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).
(129) 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).
(130) 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).
(131) 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.
(132) 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.
(133) The halogen ions Br and I can be contained singly or in combination in a concentration of 1 to 100 mM, and preferably 50 to 100 mM.
(134) It is further preferable to optionally add a reducing agent, such as vitamin C, to improve the luminescence stability.
(135) From the above study, preferable buffer formulations as a one-shot reaction solution containing a luminescent substrate specific to ALuc were narrowed down as shown below.
(136) Specifically, 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 HBSS 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, Br), and D(+)glucose or glycine.
(137) 4. Measuring Procedure and Measuring Apparatus Used in the Present Invention
(138) The ligand activity can be measured in accordance with a typical bioluminescence assay, and conventional protocols can be used without any restriction.
(139) 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 the optimum luminescent substrate of the present invention for ALuc, the luminescence intensity is immediately measured.
(140) 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.
(141) 5. Analyte of Interest in Screening Method
(142) 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. No. 6,004,617 and U.S. Pat. No. 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.
(143) 6. Kit
(144) The present invention also provides a bioassay kit comprising the luminescent substrate specific to ALuc. 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, bioluminescent enzymes, vectors comprising genes for encoding bioluminescent enzymes, cells for expressing bioluminescent enzymes, the luminescent substrate specific to ALuc according to the present invention, 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.
(145) Preferable examples of bioluminescent 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) previously developed by the present inventors. The artificial luciferases (ALuc) are particularly preferable examples.
(146) A vector comprising a gene for encoding a bioluminescent 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).
(147) Examples of control samples include positive controls comprising a bioluminescent enzyme in a predetermined amount, and negative controls not comprising a bioluminescent enzyme.
(148) 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.
(149) [III] Terms and Concepts Used in the Present Invention
(150) 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
(151) The following examples specifically describe the present invention in more detail; however, the present invention is not limited to the Examples.
(152) 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.
(153) 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: Study on Phylogenetic Characteristics and Steric Structure of Artificial Bioluminescent Enzyme
(154) Although a series of artificial bioluminescent enzymes (ALuc) were synthesized as disclosed in the patent application earlier filed by the present inventors, the details of a luminescent substrate specific to ALuc have not been unveiled. Thus, the inventors first elucidated the genetic correlation between ALuc and other bioluminescent enzymes from the phylogenetics perspective.
(155) Using CLUSTALW2.1, a multiple sequence alignment program for amino acid sequences provided by the National Center for Biotechnology Information (NCBI) in the United States, the sequence of ALuc was compared with those of Metridia longa luciferase (MLuc), Metridia pacifica luciferase 2 (MpLuc2), Gaussia luciferase (GLuc), and Lucia, which are typical marine animal-derived bioluminescent enzymes and are common in the use of coelenterazine as a substrate. The results revealed as shown in
(156) To unveil the steric structure of ALuc, the following experiment was conducted. First, the conformation of the main chain of each amino acid residue found in the X-ray crystallographic data (PDBID: 2hpsA, 2hq8A) of coelenterazine-binding protein (CBP) was converted to the corresponding codes, specifically the following three forms, -helix (h), -sheet (s), and other (o) in the manner disclosed in a related art document (Non-patent Literature 35) to describe the supersecondary structure.
(157) Subsequently, CBP and ALuc 30 were compared in terms of the homology of the amino acid sequence, and the comparison found a homology of 16.7%. Amino acid sequences were then aligned based on the homology, and amino acid residues in the structural data of 2hpsA were substituted using MolFeat v4.5 (http://www.fiatlux.co.jp/product/lifescience/molfeat/mol-index.html) to thereby prepare a molecular model. Subsequently, insertion and deletion of amino acid residues were performed using HyperProtein v1.0 (http://www.hyper.com/Products/HyperProtein/tabId/504/Default.aspx) and Chem3D Ultra v8.0 (http://www.cambridgesoft.com/Ensemble_for_Chemistry/ChemBio3D) along the alignment to achieve a supersecondary structure match, including coelenterazine. In this manner, a molecular modeling was cautiously performed. Finally, molecular mechanics (MM) calculation using the Polak-Ribiere algorithm was performed to optimize the structure.
(158) The technique described above unveiled the steric structure of ALuc30 (
Example 2: Study on Chemical Structure of Coelenterazine Derivatives
(159) Based on the steric structure of ALuc revealed by Example 1, a study was conducted to find an appropriate chemical structure of coelenterazine derivatives that matches the steric structure (
(160) Coelenterazine consists of an imidazole frame structure and three resides bound to the imidazole structure (herein, R-A, R-B, and R-C). The specificity of luciferases appears to be attributed to the structure of the residues and their functional groups.
(161) In native coelenterazine, the R-A and R-C sites have a phenol structure, and the R-B site has a benzene structure. As apparent from the steric structure shown in
Example 3: Comparison of Bioluminescence Intensity of ALuc Using Coelenterazine Derivatives
(162) To find a luminescent substrate specific to ALuc, the following experiment was conducted on the basis of the findings in the preceding Examples.
(163) First, African monkey kidney-derived culture cells COS-7 were cultured in a 96-well plate until the culture area covered 90% of the lower area of the plate. At this stage, the plate wells growing cells were divided into two groups, and pcDNA3.1(+) vector, which expresses ALuc34 or RLuc8.6-535, was introduced into each group by the lipofection technique (TransIT-LT1), followed by further incubation for 16 hours. After incubation, the cells were lysed and an aliquot of the lysates (10 L) is placed in a fresh 96-well plate. Different luminescent substrates were simultaneously added thereto using a multichannel pipette, and the relative luminescence intensity was immediately measured with a luminescence imaging analyzer LAS-4000 (FujiFilm).
(164) Luminescent substrates having a residue other than a benzene ring at the R-B site exhibited typically weak luminescence intensity and selectivity (e.g., comparison between CTZ ip, CTZ cp, CTZ fcp, and CTZ hcp).
(165) To demonstrate that the R-C site is preferably phenol (hydroxybenzene) (the speculation made in the preceding Examples), CTZ400A and CTZ h were compared in luminescence intensity. The comparison revealed that when the R-C site did not have phenol (hydroxybenzene) (i.e., CTZ400A), luminescence intensity was almost not observed at all, which compares with the strong luminescence observed when the R-C site had phenol (hydroxybenzene) (i.e., CTZ h).
(166) The experimental results indicate that the optimum luminescent substrate for ALuc preferably has a benzene ring structure at the R-A site, carrying an appropriate size of a functional group, more preferably a benzene ring structure having a halogen ion as a functional group, and particularly more preferably a benzene ring structure having iodine, fluorine, or chlorine. For R-B, a benzene ring structure is preferable. For R-C, a benzene ring residue having a hydrophilic functional group (e.g., hydroxyl group, and thiol group) is preferable, and a phenol (hydroxybenzene) structure is more preferable.
(167) The bioluminescence spectrum of a cell solution (lysate) prepared in the same manner as in Example 3A was studied. First, 5 L of the lysate was mixed with 30 L of the luminescent substrate shown in
(168) The spectrum analysis revealed that RLuc8.6-535 emits light at a wavelength that is red-shifted for about 30 nm, compared to ALuc34 when the same luminescent substrate is used. The analysis also revealed that ALuc34 emits light at a wavelength slightly more red-shifted when CTZ i is used than when CTZ f is used (Table 2).
(169) TABLE-US-00001 TABLE 1 Comparison in Luminescence Intensity RLuc8.6-535 ALuc34 Ratios ave SD ave SD (A34/RLuc8.6-535) CTZ ip 2200 969 8379 2833 4 CTZ i 97 31 913540 99031 9398 CTZ f 7151 931 956530 186595 134 CTZ op 5354 567 40564 9739 8 CTZ fcp 1217 501 3712 2349 3 CTZ hcp 2058 616 5191 1379 3 CTZ h 12812 1481 1168658 216208 91 CTZ n 2132 635 172323 28977 81 CTZ 400 3 16 11624 2965 3610 RLU/g/sec/mm2 RLU/g/sec/mm2
(170) TABLE-US-00002 TABLE 2 Comparison in Spectrum Peak ALuc34 RLuc8.6-535 CTZ f 515 547 CTZ h 519 545 CTZ i 526 CTZ ip 535 wavelength, nm
Example 4: Study on Correlation Between ALuc Substrate Selectivity and Steric Structure
(171) Typically, bioluminescence is produced by luciferase-catalyzed oxidation of a luciferin. While a wide variety of luciferases are known, the chemical structure of luciferins is not so diverse. The number of luciferins used for marine animal-derived bioluminescent enzymes is also quite limited, and typical examples of such luciferins include coelenterazine and Cypridina luciferin. In particular, coelenterazine is commonly used for many marine animal-derived bioluminescent enzymes, and thus to date about 50 or more coelenterazine derivatives have been synthesized (Non-patent Literature 30 and 31).
(172) Coelenterazine is composed of an imidazole frame structure and three residues bound to the frame structure (R-A, R-B, and R-C). The structure of each residue and its functional group are considered to be attributable to luciferase specificity.
(173) Native coelenterazine has a phenol structure in the R-A and R-C sites, with a benzene structure at the R-B site (
(174) Iodine is typically an element that is less frequently present in protein in living organisms and is also an electron donor. This fact strongly suggests that the surprising bioluminescent enzyme selectivity exhibited by CTZ i is due not only to the size effect but also the suitable interaction between iodine as an electron donor and amino acids in ALuc.
Example 5: Luminescence Detection by Dual Assay in the Presence of Both ALuc and Cypridina Luciferase (CLuc)
(175) It is advantageous for simultaneous measurement of multiple biomarkers if a dual assay can be performed in the presence of two or more bioluminescent enzymes. With this point in mind, luminescence was measured in the presence of two bioluminescent enzymes (ALuc and CLuc) (
(176) Both CLuc and ALuc exhibited high luminescence intensity in the absence of 0.1% SDS (
INDUSTRIAL APPLICABILITY
(177) By using the present invention for ligand measurement based on a reporter-gene assay, which has hitherto been widely used, or as a luminescent substrate exclusively used for ALuc 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.