TWI239353B - Thermostable luciferase mutant enzymes, nucleic acids encoding them and the use thereof - Google Patents

Abstract

A recombinant protein having luciferase activity and at least 60% similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 in Photinus pyralis luciferase is changed from an acidic amino acid to a non-acidic amino acid and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50 nm) wavelength shift in emitted light and have good thermostability. The resultant color shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.

Description

1239353 IX. Description of the invention: The present invention relates to a new enzyme, and in particular to a variant luciferase. Compared with natural enzymes, it has special properties. For DNA-encoded proteins, these enzymes can be used for analysis. About testing tools that contain these enzymes. Firefly luciferin catalyzes the oxidation of luciferin present in ATP, Mg2 +, and oxygen molecules to produce light. The quantum yield of this reaction is about 0.88. Its light-emitting properties make it widely used in photometric analysis. The amount of ATP can be measured. Examples of analysis of enzymes can be found in European patents EP-B-680515 and WO 96/02665, but other examples are commonly used in laboratories. Fluorescein can be obtained directly from insects, especially beetles such as firefly or glow-worm. Special species that can obtain fluorescein include GENJI or KEIKE fireHies, cruciata and Lucioh! Atera ! is, Donghuan's camp fire bug i (i) jef \ y) Luciola, North American firefly PAot / nus pyralfs and glow-worm Lampyris noctiluca. Anyway, because in many genes that make up these enzymes It has been copied and sorted, so these enzymes can also be aligned using recombinant DNA technology. The enzymes encoded by the recombined DNA array can be used to mutate microorganisms, such as wan coli, to produce the desired enzyme product. When analyzed in a laboratory, the light emitted by these enzymes is very similar. If the wavelength can be changed, it will make it easier to read special detectors, or it will help to use in systems that require multiple loggers. For example, 1239353 is used to monitor different events in the same sample. One method to identify record molecules is to use fluorescent molecules that emit light at specific wavelengths. This method can be achieved by using fluorescein-containing record molecules obtained from different species of beetles or fireflies. A feasible method is to use recombinant DNA Technology to make mutated fluorescein to make changes in the signal wavelength. Examples of such mutations can be found in patent WO 95/18853. In addition, the thermal stability of natural and recombinant forms of luciferin is that luciferin will quickly lose its activity when exposed to temperatures above 30 ° C, especially above 35 ° C. Instability can cause problems when used and stored in high temperature environments, or if analysis must be performed under high temperature reaction conditions, such as to increase reaction speed, problems can also occur. It is known from patents EP-A-524448 and WO 95/25798 that mutated luciferin has improved thermal stability. In these descriptions, it is first mentioned that there is a mutated 217 in Japanese firefly luciferin. Variant luciferin, especially using an isoleucibe fragment to replace the three-hydroxyline (threonine) fragment, and then mention to the variant phytofluorescein and self-reliance ^ pyralis, Luciola mingralica 'Luciola cruciata Compared with the luciferin obtained by Luciola, it has more than 60% similarity, but the amino acid fragment corresponding to the valence fragment 354 or the type 356 fragment is mutated to be different from glutamate, and it is particularly It is different from glutamate, aspartate, proline or glycine. The co-filed British patent application number 9823468.5, as well as the international patent application derived from this application, mentions more of these types of variations, among which 1233353 mentions proteins, which have fluorescent activity and are natural Luciferin, Photinus pyralis 'Luciola mingralica' Luciola cruciata or / WWZ? Enzymes, have at least 60% similarity, but there are variations in various positions of the protein, including (30 street should be PAoijfnus pyraiis Fragment 214 of Luciferin and the amino acid fragment of LucioJa mingralica 'Luciola cruciata or Luciola lateralis 6; or (b) a 232 fragment of Luciferin that is compatible with Photinus pyraiis and LucioJa mingralica ^ Luciola cruciata WL Luciola lateralis 234 fragment of the amino acid fragment; or (c) an amino acid fragment of the luciferin fragment 295 忤 of PJiotinus pyrahs and the LucioJa mingralica 'Luciola cruciata ^ Luciola lateralis 297 fragment. These applications find differences in luciferin Changing (or adding) amino acids in position can make the wavelength of the emitted light very different And / or can improve the thermal stability of the enzyme. In addition, it can improve the proton flux of the emitted light, making the enzyme more suitable for living organisms (//? Κ / κο) Analysis, or suitable for use in test tubes without CoA or other 'luminodynamic sensing' compounds ("P7Υ / Ό). The present invention provides a recombinant protein with fluorescent activity and at least 60% of natural fluorescein Similarity, which sequentially constitutes an enzyme. Compared to the natural luciferin of the tree, the amino acid fragments of the 357 pairs of fluorescein that correspond to PAotimis PyrWs have variations, so that the fluorescent enzyme is compared with the natural luciferin. It can emit light of different wavelengths, and / or can increase thermal stability compared to natural luciferin. 1239353. Natural luciferin sequences can be used as the main component of the recombinant form of the present invention, including Photinus pyralis, Luciola mingralica, Luciola cruciata ^ Luciola lateralis 'Hot aria parvula, Pyrophorus plagiophthalamus Lampyris noctiluca' Pyrocoelia mivako, Photuris pennsylvanica or PAr / zc ^ Ar / Xrailroad-worm — See Biochem. 38 (1999) 8271-8279). Bioluminescent enzymes obtained from species can use matrix D-luciferin (4,5-dihydro-2- [6-hydroxy-2-benzothiazolyl] -4-thiazol carboxylic acid) to emit light. Pentabioluminescent enzymes can be used as The main component of the variant enzyme of the present invention. Specific natural photoninogen sequences that can be the main component of the recombinant form of the present invention include Photinus pyralis, Luciola mingralica, Luciola cruciata Luciola lateralis' Hotaria parvula, Pyrophorus plagiophthalamus Lampyris noctiluca 'Pyrocoelia mivako' 特别 ^ × and Photuris penylvanylvanyl The thing is, luciferin is a waste element that can be extracted from mingralica, Luciola cruciata or Luciola lateralis. In Luciola mingralica, Luciola cruciata, or Luciola enzymes, the appropriate amino acid fragment is at position 359 in the sequence. The sequence of all kinds of luciferin shows that these luciferins have a very important demarcation between their similarities, which means that most of the similar regions can be detected by sequence inspection, and it can be easily determined in these enzyme sequences 1239353 corresponding regions, although under certain conditions, in order to determine the corresponding regions or specific amino acids between various sequences, commercial software must be used (such as "Bestfit" provided by the University of Wisconsin Genetics Computer Group; (See Nucleic Acid Research 12 · 387-395 of Devereux et al. (1984)). Alternatively or in addition, the corresponding amino acid can be determined by referring to BioChim · Biophys Acta 1339 (1997) 39-52 of L. Ye et al., Whose enzyme sequence is shown in the works, including numbering, and its numbering system will be linked to this application . Regarding possible changes in the amino acid fragment corresponding to fragment 357 in luciferin, most natural sequences will have an amino acid fragment (aspartic acid or glutamic acid) at this position. ), For example, f is in some types of luciferin, in which the corresponding fragment (355) is valine, which is a non-polar fragment, or in some types of luciferin Its corresponding position is V354 in Pvc} r or PhRE, which is the leucine of L354, so it is usually used at this position as the substitution amino acid succinic acid, glutamic acid, valeric acid or leucine Other amino acids. Therefore, in most cases, amino acid fragments are replaced with fragments that are not acidic, including basic amino acids like lysine or argimne, and non-polar amino acids like leucine , Valerate or isoleucine, uncharged polar amino acids like tyrosine, asparagine, glutamic acid, phenylalanine, serine, chromosomal amino acid ( tryptophan) or hydroxybutyric acid. And especially substituted with uncharged polar amino acids, such as tyrosine, aspartin, silk amino acids or hydroxybutyric acid 1239353, where the preferred amino acid fragments for substitution at this position are tyrosine, phenyl Alanine or tryptophan, and most preferably tyrosine. In general, aromatic fragments can increase the maximum displacement at this position and can contribute to thermal stability. The natural sequence includes a non-acidic amino acid fragment at this position, which can be mutated into different non-acidic fragments. Using this method to change the enzyme can be found that the wavelength of the light emitted by luciferin is changed, and in some cases the wavelength can be shifted by 50 nm toward the red end of the spectrum. Therefore, the part of D357Y fluorescein fluorescein emits light at a wavelength of 612 nm. Compared with natural enzymes, the wavelength of light emitted is 562 nm. The 50nm wavelength shift has a negligible effect on analytical applications, and when the amount of this shift can be easily defined on the spectrum. Different colors of fluorescein can be used as marker molecules in gene performance studies, and more than one gene can be monitored at the same time, such as described in WO 95/18853. Multiple analysis tests can also be performed with luciferin as a marker . In fact, the color of the light in this case is deep red, which is particularly useful in analytical methodologies. Variations in red can be useful when analyzing an ATP solution containing pigments or other compounds that absorb light called short wavelengths. For example, the red solution does not absorb red light. The red solution. Examples of objects often used for such analysis include blood samples or solutions of eukaryotic cell culture media, which may contain a Red pH indicator. The ability to generate a crimson signal is useful when using a colorimetric reagent such as a mixture of fluorescein 11 1239353, especially when another reagent in the sample produces a green signal in the photocathode spectrum The photomultiplier tube used in the analysis can be set to detect one or two peaks generated in the signal sample. In other words, the red and green radions can be distinguished in the same sample. Photon magnetic force. In addition, it can be found that the wavelength shift can be affected by the presence of cofactor CoA (CoA). This feature increases the possibility of applying this enzyme to the analysis of cofactors. As described below, the effect of cofactor A on the emission spectrum in the test tube has been carefully examined. The concentration of coA increases the change in the spectral distribution, and at the highest concentration of CoA, it passes through the 590-630nm region. In between, a wavelength with a peak at 610nm can control the spectrum. Therefore, according to another aspect of the present invention, there is provided an analysis method for detecting the presence of CoA in a sample, wherein the analysis method includes adding a sample that may contain CoA, including the above-mentioned fluorescer, and used to generate fluorescence (luciferase / lucufenm) other reagents required for the reaction; measuring the wavelength of light emitted from the sample; correlating the results with the presence or absence of CoA. Such analysis can be useful in the detection of growing or viable cells, such as in microorganisms or eukaryotic cells. For example, the concentration of CoA in K cells is quite high and has a good effect on metabolism. The variant enzyme of the present invention can be used to monitor the state of metabolism in the organism, especially the concentration of CoA in the living body, because the light is emitted The change in wavelength is related to the concentration of CoA. When manufacturing antibiotics (such as in streptomycetes), CoA is an important main 12 1239353 metabolite, in which case the above analysis method is particularly useful; the concentration of CoA in the cell is also an important fatty acid biosynthesis Indicator, and there will be changes when the cells are starved. Some metabolic disorders, such as carcinogenesis and diabetes, will have abnormalities in fatty acid metabolism, resulting in abnormal amounts of CoA. The present invention Analysis can be used to diagnose these situations. For example, in a cell sample, such as a blood sample from a patient, the amount of CoA can be determined during the analysis by measuring the wavelength of light emitted by a luciferin of the present invention. This result can be compared with a Samples obtained from healthy cells are compared to determine if there is a change in wavelength, and therefore there is a corrected amount of CoA, which can be used to show the patient's condition. Prior to analysis, cells are disrupted and lysed using a known cytolytic agent. It is generally considered that the amino acid fragment at position 357 must be connected to the junction of co-A, and when the surface of the luciferase is drawn with a resolution of 1 angstrom (using the SYBL protein model software from Tripos Ltd.) A small polar opening will be marked. This opening is underlined as fragments H310, E354, and D357, and is measured between 8-10 angstroms. Viewed from the top of the molecule, this opening is like a part of a larger opening, marked by H310, E354, D357, and 1232. The fragments H310 and E354 are used as one across two smaller A bridge with an open appearance of cracks (as shown in Figure 8). There is no need to use the principle to connect. When the enzyme is placed in the solution to provide a larger opening to allow CoA to connect (a depth of about 12 Angstroms and a width of about 8 Angstroms), the bridged fragments may have sufficient flexibility to detach, Consistent with the results of the energy calculation. 13 1239353 When five c0 / y cells expressing the firefly luciferin variant of the present invention grow on different carbon raw materials, a change in the radiation light spectrum in the living body can be observed; acetate or glucose is used as the carbon The change of raw material from a catalyst-rich (LB) to a minimum defined catalyst will shift the emitted light to a longer wavelength and reduce the contribution of a shorter wavelength. This may be used as a control in analysis. A further method of radiating the wavelength of light. A mutation at position 357 of the protein has been found to cause an increase in thermal stability. When the protein's luciferin activity is not excessively compromised, proteins with further mutations can be provided. These mutations are suitable to improve the properties of the enzyme or make it more suitable for certain purposes in certain methods. This may indicate these mutations. The result is improved thermal stability and / or color shift characteristics, and / or Km of the ATP of the enzyme. Examples of variations that enhance color shift can be found in the WO95 / 18853 patent; examples of variations that affect Km 値 are in the WO96 / 22376 patent and in the international patent application number PCT / GB98 / 01026 Mentioned in. In general, the effects of mutations have been found to be added to items with changing characteristics. The variant luciferin provided by the present invention may include other special variations. Compared with natural luciferin, these variations can improve thermal stability, especially including at least one of the following situations: (a) Corresponding to fluorescence The amino acid fragment of amino acid 354 (or 356 in luciferin) of the mutin has a variation; 0 >) The amino acid fragment corresponding to the position 215 of the PJiodmis campionin (217 in the LucioJa campionin 12393353) is a different Hydrophobic amino acid; or (c) an amino acid fragment corresponding to fragment 214 of lumenol, or an amino acid fragment corresponding to fragment 216 of Luciola mingrelica 'Luciola cruciata ^ Luciola lateralis; (d) corresponding to valence fluorescence Fragment 232, or an amino acid fragment corresponding to fragment 234 of Luciola mingrelica 'Luciola cruciata M Luciola lateralis; (e) Amino acid corresponding to fragment 295 of luciferin, or a corresponding, to Luciola mingrelica, Luciola cruciata or Luciola lateralis luciferin fragment 297 amino acid fragment; (f) corresponds to amino acid 14 of / ¾luciferin, or corresponds to LucioJa mingreh The steam segment 16 of ca, or the amino acid fragment of fragment 17, LucioJa cruoiata or fragment 17; (g) Corresponding to the amino acid 35 of ^ / 7 luciferin-like, or the segment of Luc] 'oL · mingrehca 3Ί, or Fuying shoulder, Lucioh cruciata or amino acid fragment of fragment 38; (h) amino acid fragment 105 corresponding to valence luciferin, or fragment 106 corresponding to Z ^ C / o / 3, or Amino acid fragment corresponding to fragment 107 or gene fragment 108; ⑴ amino acid fragment 234 corresponding to luciferin, or amino acid fragment corresponding to rabbit Luciola mingrelica, Luciola cruciata and Luciola lateralis 236; 15 1239353 (j) corresponds to Amino acid fragment 420 of luciferin, or an amino acid fragment corresponding to 3 \ Luciola mingrelica 'Luciola cruciata M Luciola lateralis 6¾ fragment 422; (k) amino acid fragment 310 corresponding to luciferin, or 3 \ Luciola mingrelica' Luciola cruciata M Luciola lateralis fragment 312 amino acid fragment; the fragments corresponding to the amino acids appearing in the sequence of the natural species are different, and compared to these Natural amino acid has the opposite luciferin, luciferase these enzymes to enhance thermal stability. Therefore, the preferred embodiment of the protein mentioned in the present invention is a mutated natural luciferin, in which more than one amino acid is not the same as the amino acid at a relative position in a suitable natural enzyme, and the number of amino acids such as 100 amino acid fragments, preferably no more than 40 amino acids, and most preferably up to 30 amino acids. Therefore, in a preferred embodiment, the protein of the present invention includes luciferin, in addition to the above-mentioned variation at position 357, it also includes at least one of the following: a) amino acid 354 corresponding to P-force luciferin Is an amino acid fragment other than glutamate; b) the amino acid fragment corresponding to position 215 in py / Ws luciferin is a water-repellent amino acid other than alanine; c) corresponds to the amino acid fragment in luciferin Position 214 is an amino acid fragment other than butine; d) The amino acid fragment corresponding to position 232 in luciferin is the amino acid 12393353 of isoleucine; e) The position corresponding to position 295 in luciferin is other than phenylalanine F) Corresponding to the amino acid fragment Λ ^ / ^ like pyrWs luciferin is an amino acid fragment other than phenylalanine; g) Corresponding amino acid 35 in luciferin is an amino acid fragment other than leucine H) corresponding to the amino acid fragment 105 in luciferin is an amino acid fragment other than alanine; i) corresponding to the amino acid fragment 234 in fensi / 7JTA75 luciferin is an amino acid fragment other than glutamic acid; j) Corresponds to Firefly Amino acid fragment of factors other than the 420 amino acid fragment wire acid gas; K) corresponding to the like; amino acid fragment of the luciferase is an amino acid fragment of 310 amino acids other than tissue. The protein of the present invention includes Lucj'oJa mj'ngreJica, Lucj'oJa crucj'ata or luciferase sequence [] of enzyme, in addition to the above-mentioned position 359 is mutated, at least one of the following conditions is included: The amino acid 356 corresponding to luciferin is an amino acid fragment other than glutamate; b) the amino acid fragment corresponding to position 215 in luciferin is a water-repellent amino acid other than alanine and hydroxybutyric acid;

Luciola mingrelica 'Luciola cruciata Luciola h such as fluorescein fragment 216 is an amino acid fragment other than aminoacetic acid (for his sequence consisting of 17 1239353), or aspartin (for Luciola cruciata or Luciola lateralis sensitive sequence) Amino acid fragments from Js; phantom lines, fragments 234 in Luciola mingrelica, Luciola cruciata, or Luciola luciferin are amino acid fragments other than silk amino acids; &) are in LucioL · mingrelica, Luciola cruciata, or Luciola luciferin Fragment 297 is an amino acid fragment other than leucine; f) the amino acid 17 of Luciola mingreiica, Luci'oh c / T / c / a々, or fluorescein is Amino acid fragments other than phenylalanine; g) is a fragment of Luciola mingrelica Υ! Or: LucioL · cruciata or fragment 38 is an amino acid fragment other than lysine; h) corresponds to Zi / c / oAg amino acid fragment 106, marginal amino acid fragment 107, or / amino acid fragment 108 are amino acid fragments other than aminoacetic acid;

Luciola mingrelica 'Luciola cruciata Luciola's amino acid fragment 236 is an amino acid fragment other than glycine; Luciola mingrelica, Luciola cruciata or Luciola's fragment 422 is an amino acid fragment other than hydroxybutyric acid;

Luciola mingrelica 'Luciola cruciata or Luciola's amino acid fragment 312 is an amino acid fragment other than butine (for the sequence consisting of / 27 // 7 ^ re / i ^ 2), or valeramic acid (for , Lucioia cruciata or LucwJa iaterahs). In any case that helps to improve thermal stability, the specific substituted amino acid can be determined by the standard methods mentioned later. In each case, different substitutions may improve thermal stability. Substitutions can be improved by Position-directed mutations in the nature of DNA coding, or the appropriate mutated proteins known to those skilled in the art, will affect this. In this example, the present invention combines the identification of positions, where positions are associated with thermal stability. In general, it is always thought of replacing with an amino acid with different characteristics compared to the natural amino acid. Therefore, a hydrophilic amino acid fragment may be replaced with a hydrophobic amino acid fragment in some cases, and vice versa. The acidic amino acid fragment can be replaced with a basic fragment. For example, a protein may include a fluorescently active protein, i and Photinus pyralis, Luciola mingrelica, Luciola cruciata, or an enzyme have at least 60% similarity in the sequence of the enzyme, including at least one of the following: a) corresponding There is a variation in the amino acid fragment of Hujiata ’s py / Ws luciferin fragment 214, or the fragment 216 corresponding to Luciola mingrelica 'Luciola cruciata M LucioJa lateralis, and in the case of luciferin, hydroxybutyronine Other amino acid fragments; or b) corresponds to fragment 232 in luciferin, or corresponds to fragment 234 of Luciola mingrelica 'Luciola cruciata Luciola lateralis, and the amino acid fragment of 234 is mutated, and in the case of luciferin, it is opaque Amino acid fragments other than amino acids; or 19 1239353 C) Corresponds to fragment 295 in fluorescein fluorescein, or corresponds to Luciola mingrelica 'Luciola cruciata Luciola lateralis 297. The amino acid fragment has variations, and for example in ΑΚα / Α Examples of luciferin are amino acid fragments other than phenylalanine; and compared to natural luciferin, These fluorescent enzyme having enhanced thermal stability. The sequence of all kinds of luciferin shows that these luciferins have a high degree of similarity between them, which means that by detecting the most similar regions to check their sequences, it is easy to determine the corresponding regions in these enzyme sequences. Although commercial software may be used (such as 'Bestfit' provided by the University of Wisconsin Genetics Computer Group, to determine specific regions or specific amino acids between various sequences; see Nucleic Acid by Devereux et al., 1984 Research 12: 387-395), or the corresponding acid can be determined with reference to Biochim. Biophys Acta 1339 (1997) 39-52 of L. Ye et al. With regard to the amino acid fragment corresponding to fragment 214 of luciferin The polar amino acid hydroxybutyrate is suitable to be replaced by a non-polar amino acid, such as alanine, glycine, valerate, leucine, isoleucine, proline, phenylalanine, methionine, Tryptophan, or cysteine. The hydroxybutyric acid fragment used to replace the fragment 214 corresponding to the amino acid VII is preferably alanine and more preferably cysteine In any case, different polar fragments at this position, such as aspartic acid, can also increase the thermal stability of the corresponding enzyme with hydroxybutine at this position; in the natural fluorescent enzyme, Other amino 20 1239353 forgings in this position include gas-based forging {Luciola mingrelica, Hotaria parvula ,, aspartin {Pyrophorus plagiophthalamus, GR, YG, YE or 〇, Luciola Cruciata 'Luciola lateralis' Lampyris noctiluca' Pyrocoelia Miyako Photuris pennsylvanica LY, KW, J19) VXSl silk amino acids, these are replaced by non-polar or different non-polar side chains, such as alanine and cysteine. Compared to valence luciferin In the possible changes of the amino acid fragment corresponding to fragment 232, the non-polar amino acid, isoleucine, is suitable for being a unique non-polar amino acid, such as alanine, glycine, valerate, leucine, Proline, phenylalanine, methionine, tryptophan, or cysteine, other amino acids that occur at this position in the natural sequence Include silk amino acids and aspartin. It should be used in /% <? ///? IA? Campionin, in the steam section 2Ύ1, and in Luciola mingrelica, Luciola or / WWh luciferin, it corresponds to fragment 234. The preferred amino acid fragment for substitution is alanine. Changing the amino acid fragment corresponding to fragment 295 in Yangjia's "fluorescein-like" and corresponding to fragment 297 in Yang 6 ^ / 7 seems to still be Yang //? Luciferin can also affect the thermal stability of the protein (in Fluorescein corresponds to position 292), usually the amino acid at this position is a non-polar amino acid, phenylalanine or leucine. Substitution with a unique non-polar amino acid is appropriate, for example. Say, in / ¾ such as · 77M valence luciferin, the non-polar amino acid, phenylalanine, is suitable to be replaced by a non-polar amino acid, such as alanine, leucine, glycine, valerate , Isoleucine, lactate, methionine, tryptophan, or cysteine, 21 1239353 is a better amino acid for replacing phenyl phenylalanine in fragment 214 in luciferin It is leucine. It is added to the amino acid η corresponding to the luciferin, or the amino acid 16 or 17 corresponding to hc / 仏 fluorescein (in Yang / 7 > 〇 your 7 · ζfluorescein is 13) The amino acid fragment may also be mutated. This amino acid fragment (usually phenylalanine, but can also be white Acid, silk amino acid, arginine, or tyrosine in some examples) are suitable for different amino acids, especially a unique non-polar amino acid, such as alanine, valeric acid, leucine, Isoleucine, proline, methionine, or tryptophan, preferably alanine. Amino acid 35 corresponding to PAot / mJspjrWs luciferin, or amino acid 37 corresponding to 1-oh luciferin (in other families Variations of the amino acid fragment of 38) are also useful. Various amino acids at this position of this natural enzyme include leucine but can also be lysine, tissue amino acid, aminoacetic acid, alanine, glutamic acid, And aspartic acid, the amino acid fragment at this position is suitable for a non-polar amino acid fragment or an amino acid with different aircraft properties, such as alanine, valerate, phenylalanine, isoleucine, Proline, methionine, or tryptophan to replace, the best amino acid at this position is alanine, which is different from natural enzymes. The amino acid corresponding to position 14 of the sequence, and / or corresponds to w-valent fluorescent Photoammonia The variation of the amino acid fragment of the amino acid 35 is preferably not only a mutation in the enzyme, but also suitable for the other mutations defined above, especially at the position corresponding to the position of P plus fen μ luciferin 22 1239353 214, 395, or 232. Variations in location. The street should be PAotinus pyrai / s fluorescein fragment Λ05, and the corresponding Luciola mingrelica campion fragment, Luciola cruciata campion fragment 107, or ππ / σΑ crowdification / your luciferin fragment 108 ( In 102, the change of the amino acid fragment will also affect the thermal stability of the protein. Usually the amino acid at this position is a non-polar amino acid, alanine or aminoacetic acid, or a silk amino acid (in), suitable for Use a non-polar amino acid to change, for example, in the non-polar amino acid, alanine, it is suitable to use a unique non-polar amino acid, such as phenylalanine, leucine, glycine, valerian Amino acid, isoleucine, proline, methionine or tryptophan acid, among which the amino acid fragment that is more suitable for replacing the alanine fragment corresponding to the luciferin fragment 105 is valerian Acid. Changes in the amino acid fragment corresponding to the 234-like luciferin fragment and Luciola mingrelica 'Luciola cruciata ^ Luciola laterlis fragment 236 (in most cases, 231) will also affect the thermal stability of the protein, usually in The amino acid at this position is aspartic acid or glycine, and in some cases glutamic acid or hydroxybutyric acid, where it is appropriate to change with a non-polar or unique non-polar amino acid, for example, in In addition, the aspartic acid amino acid fragment is suitable for a non-polar amino acid, such as alanine, leucine, glycine, valeric acid, isoleucine, proline, methionine or tryptophan. The amino acid fragment corresponding to the phenylalanine fragment corresponding to the / ¾⑽ luciferin fragment 234 is 23 1239353 aminoacetic acid; the non-polar amino acid fragment at this position (in Zz / c / oh fluorescein as an example), such as glycine can be replaced with a special non-polar amino acid. Changes in the 420-luciferin fragment and Luciola mingrelica 'Luciola cruciata ^ Luciola laterlis-fragment 422 (417 in green and 418 in red) changes in the amino acid fragment will also affect the thermal stability of the protein The amino acid at this position is usually an uncharged amino acid, silk amino acid, hydroxybutyric acid, or aminoacetic acid. It is suitable to be changed by a special uncharged amino acid. For example, in Naiga / λμ Silk amino acids can be substituted with aspartic acid, glutamic acid, hydroxybutyric acid or tyrosine, and especially hydroxybutyric acid. The change in the amino acid fragment of the fragment 310 corresponding to PAodnus py / ^ // s luciferin, and the fragment 312 corresponding to Luciola mingrelica 'Luciola cruciata ^ L Luciola laterlis also affects the thermal stability of the protein. Among the proteins, the amino acid regularization at this position is tissue-oxygen in a fish form of Photinus pyralis, Pyrocoelia miyako, Lampyris noctihica, and Photuris pennsyhnvanica, and in Lucioia mingrelica, Hotaria parvula, and amino acids 307) Hydroxybutine in luciferin, valerate in and

Py / OpArow ρ / sigh / spot price / triple luciferin is aspart. Therefore, the amino acid at this position is usually a hydrophilic amino acid. It can be changed by adding special amino acid fragments that increase the thermal stability of the enzyme. It is especially used to replace tissue amino acid fragments corresponding to fragment 310 in 24 1239353 fluorescein. Suitable is arginine. Other mutations may also be present in the enzyme. For example, in a preferred embodiment, the protein may also be placed at the corresponding position with the "fluorescein-like amino acid 354 (or 356 in luciferin)". Altered by amino acids of glutamic acid, especially amino acids other than aminoacetic acid, proline or aspartic acid, suitable amino acids at this position are tryptophan, valeric acid, leucine, isoleucine Or aspartic acid, but more suitably lysine or arginine, this variation is described in WO 95/25798. It can be found that the water-repellent fragment at this position will increase the wavelength shift of the enzyme, In addition, the presence of a large water-repellent (V or I), polar (N), or positively charged (K or R) amino acid at position 354 will improve thermal stability. In an alternative preferred embodiment The protein also has a position corresponding to the amino acid 217 of the amino acid 217 in Luciola luciferin (in the case of 215) is changed to a water-repellent amino acid, especially isoleucine, leucine or valeric acid, such as EPP-A-052448. Proteins in the invention Including both natural and recombinant fluorescent enzymes, which are at least 60% similar to the natural sequence, such as enzymes such as μ pyralis, Luciola mingrelica, Luciola cruciata, or Luciola enzymes. It is understood that at least 60% of the natural enzymes are Amino acids are present in the protein of the present invention. Such proteins may have a high similarity, especially at least 70%, preferably at least 80%, and more than 90% of the natural enzymes listed above. Similar proteins include allelic variants, proteins from other insect species' like 25 1239353 recombinantly produced enzymes. It can be easily confirmed that these proteins are encoded by nucleic acid These nucleic acids are mixed with a sequence of natural enzymes under stringent hybridization conditions. Among them, those skilled in the art can easily understand the conditions referred to, and they can be used in Molecular by Sambrook et al., 1989. The example given in Cloning (Cold Spring Harbor Laboratory Press) serves as an example. In general, low stringency conditions can be defined Three times the SSC at 65 degrees Celsius, and 0.1 times the SSC at 65 degrees Celsius under severe conditions, where SSC is the name of the buffer solution, including 0.15M sodium chloride, 0.015M Trisodium citrate, three times the SSC means three times as strong as the SSC. In particular, the similarity of a specific sequence of the sequence of the present invention can be evaluated by multiple alignments. Proposed by Lipman and Pearson (Lipman, DJ. &Amp; Pearson, WR · (1985) Rapid and Sensitive Protein Similarity, Science, vol 227, ppl435-1441). The "optimistic" percentage range should be calculated using the following parameters for the Lipman-Pearson algorithm: ktup = 1, gap penalty = 4 and gap penatly length = 12, and sequences evaluated for similarity should be used as "Test sequence", which means that it is a basic sequence for comparison, such as the sequence of AKra / Zi? Or any of the other sequences recorded by the Ye Temple people, can be brought into the above algorithm. A specific example of a protein of the present invention is a natural luciferin sequence having one or more mutations, as described above. The present invention further provides a nucleic acid compiled into the above-mentioned luciferin, wherein the nuclear 26 1239353 acid system is a conventional natural The basics of the sequence are based on the knowledge of the genetic code, and appropriate mutations will cause the expected changes in the amino acid sequence to be easily expressed. In a preferred embodiment of the invention, the nucleic acid is a synthetic gene, and the synthetic gene is designed to Remove the codons found only in important expressed genes. These dominant genes are taken from a common genetic trait host like five cW /, and At the same time, it is possible to avoid the introduction of the cryptographic unit found only in the beetle's gene coding. This method can ensure that the gene has a cryptographic unit, which is applicable to the performance system of five insects. For example, whether it is used for Amino acid code unit, whether arg, leu, ile, gly, and pro may be changed to CGT or CGC (arg), CTG, CTT or CTC (leu), ATC or ATT (ile), GGT or GGC (gly), and CCG, CCA, or CCT (pro) will therefore delete the rare code units. In the following description and the example of the synthetic gene illustrated in Figure 14, a total of 139 stationary mutations result in 62 new non-rare (non- rare), a cryptographic unit (11% of the total), at the ribosome junctions of the first 8 nucleotides forming part shown in Figure 14, so no code is specified, and the coding sequence is a methionine fragment indicated by an upward arrow Initially, the coding sequence is similar to a similar sequence in a preferred embodiment of the present invention, such as a sequence having at least 90% similarity, or a sequence having at least 95% similarity.

Another useful feature is the combination of new unique defined positions, which can be used to generate a synthetic set, these positions are simpler and more efficient to generate mutant genes on genes, especially combined cassette mutant genes ( cassette mutagenesis), which is particularly suitable for creating unique defined positions between the cDNA codes of the subdomain B 27 1239353 in the enzyme, and additionally generates a unique defined position at the end 3 of the gene, which helps to allow simple Fusing, and / or removing peroxy obscure target sequences. In the examples introduced hereafter, nine new uniquely defined positions are counted, most of which are in the middle third position of the gene, and a unique last three positions are formed at the endpoint 3 'of the gene. To allow simple C-terminal kiln-occupy fusion (see Figure 12). Finally, the use of synthetic genes can be used to generate mutations to increase the thermal stability of the gene product, or to modify the properties of its products in other ways. In the lines mentioned below, three non-stationary mutations are used to generate The thermostable amino acids convert T214C, E354K and D357F into polypeptide ° The nucleic acid of the present invention is suitable for binding with a dominant vector, such as in a control unit, such as a promoter, a promoter (enhancer), terminator (terminator) and other cytoplasmic bodies, these vectors can then be used to deliver a host cell, such as a prokaryotic or eukaryotic cell like a plant or animal cell, especially in A prokaryotic cell appears to be ugly, so the cell can exhibit the characteristics of a desired fluorescent enzyme. 'Transferring cell cultures like this results in the production of fluorescent enzymes, and the conditions for their use are known to those skilled in the art. These fluorescent enzymes It can then be isolated from the culture medium. When the cells are plant or animal cells, these cells can be used to propagate plants or animals. In order to extract proteins from these plants, or in genetically modified animals, proteins can be retrieved from milk, as vectors, transformed cells, genetically modified plants and animals, and methods for producing enzymes by culturing these cells. Both are included in a further object of the present invention. The following describes the use of random mutated fluorescein. Among them, it is found that the single point mutation of D357Y produces a large color shift in the wavelength of the emitted light. Compared with natural luciferin, it also has a very large color shift. Large thermal stability, further investigation shows that substitution in a certain range at this position can be improved to a good thermal stability, and / or there is a large color shift. Specific examples of Fen variant enzymes that fall within the scope of the present invention are as follows:

D357Y

D357F

D357W

D357K

D357N

D357I

E354I / D357Y

E354V / D357Y

E354C / D357Y

E354R / D357Y

E354S / D357Y

E354N / D357Y

E354K / D357M

E354R / D357L 29 1239353

E354W / D357W

E354H / D357W

E354R / D357F

E354K / D357F

E354S / D357F

E354M / D357F

E354A / D357R

E354A / D357F

E354T / D357Y

E354A / D357N

I351M / E354R / D357V

E354S / D357V

E354R / D357W

E354R / D357M

E354R / D357S

E354N / D357S or equivalent to any of these when taken from luciferin from other insects. Using the localized mutant gene (PCR) or a combined box-like mutant gene, the mutation used to generate the above changes is introduced into the luciferin gene on the cytoplasmic pET23, and oligonucleotide is added to the PCR reaction. To produce correlated mutations, as described below. As mentioned previously, the effect of point-like mutations at positions 354 and 215 is additive, and the present invention provides the possibility of combining three or more mutations to provide a high level of 1239353 in a variant enzyme with a large color shift Thermal stability. The luciferin protein of the present invention is very useful in bioluminescence analysis, which uses the luciferase / luciferin reaction as a signal object. Many such analyses can be found in the literature, so proteins can be included in the preparation The test kits used for these analyses are selectively combined with luciferin and other reagents required for specific analyses to enable the above-mentioned objects, features, and advantages of the present invention to be achieved. It is more obvious and easy to understand. The preferred embodiments are described below in detail with the accompanying drawings as follows: A brief description of the drawings: FIG. 1 is a logarithmic chart showing the temperature at 45 ° C in the present invention. The relationship between the percentage of the remaining activity of the cultured mutant enzymes and time; Figure 2 is a spectrum diagram obtained from cultured cells with fluorescent enzymes, this cell line is in a citrate buffer solution with D-luciferin, The enzymes used are (a) recombined natural Fan (9-valent flea light, (b) a variant of D357K, (c) a variant of D357N, (d) a variant of D357W, (f) a D357F variants, g) —species D357Y and (h) —double variants of E354I + D357Y; Figure 3 shows the remaining activity percentage and time of three variant enzymes, E354I, D357Y, and E354I / D357Y double variant (DM). Figure 4 shows a graph of the radiation spectrum, including the recombined natural enzymes, and (b) double variation (DM) E354I / D357Y; Figure 5 is a chart of the photon radiation attenuation rate, including a reconstituted 31 1239353 Natural enzyme (♦) r-wt and a D357K variant enzyme (j); Figure 6 shows a molecular model diagram illustrating a possible CoA connection opening in a fluorescent enzyme; Figure 7 shows in a living organism, Bioluminescence spectrum emitted by Coli cells, this cell shows a variant A Aj valence D357Y (a) growing on LB; (b) growing on very small media and sodium acetate; (c) growing on very small media and Glucose grows; Figure 8 shows the bioluminescence spectrum emitted by Coli cells in a living organism, this cell exhibits a mutant family. Luciferin E354K / D357M (a) grows on LB; (b) In tiny media with sodium acetate (C) Growth on very small media and glucose; Figure 9 shows a user-friendly. The mutation of luciferin D357Y, the impact of CoA on the spectral contribution of the emitted light, Figure 10 shows a utilization A chart of standardized data for the mutation of A-valent fluorescein D357Y and the effect of CoA on the spectral contribution of radiated light; Figure 11 shows a mutation using Pura-valent fluorescein E354I / D357Y (Figure 11a). A graph of the impact of the spectral contribution and standardized data (Fig. Lib). Fig. 12 illustrates the restricted position changes used in a synthetic luciferin gene architecture. Fig. 13 illustrates the variation in a luciferin gene. The architecture used in the synthesis; Figure 14 shows the sequence of the luciferin gene cDNA (SEQ ID NO 1) (including ribosome 1-8, which is part of the ribosome junction unit but is not encoded), and the arrow pointing up The indicated methionine fragment begins to encode the amino 32 1239353 amino acid sequence (SEQ ID NO 2); and Figure 15 illustrates the thermal stability of the variant encoded by the synthetic gene at 50 ° C. The first embodiment is a variant of luciferin identification and feature identification. Prepared by the error-prone PCR [M. Fromant et al., Anal. Biochem. (1995) 224, 347-353] Two gene segments of luciferin, one of which includes the error-prone PCR product of the full-length gene, was replicated in the T7 display system pET23a (Novagen Inc., Madison, WI, USA), and the second gene segment included / An error-prone PCR product of a short fragment of the π gene, including amino acids 199-352, was replicated in the vector pBSK (+) (Stratagene, La Jolla, CA, USA). The pET23a gene segment is expressed in the five systems BL2UDE3) , {E.CoIi BY dcm ompT hsdS (rB · ιώβ ·) gal and (X > Έ3)). The pBSK (+) gene segment is expressed in HB101 cells, (aral4 galK2 lacYl A (gpt-proA) 62 rpsL20 (Strr) xyl-5 mtl-1 red / J ”thin / rC-mre valence W. pET23a and pBSK (+) Both have the gene of β-lactamase, and make the cells with cytoplasmic body resistant to ampicillin. A Wan.6 ^ // system is converted together with the gene segment prepared by electroporation and used A BIORAD five π / y pulser was grown on LB agar at 37 ° C overnight, and the ampicillin concentration of 50 μβ / πιb cells was sent to a nylon film (Osmonics, Minnetonka, Minnesota, 33 1239353 USA ·), and sprayed with a luciferin solution (500 μM D-luciferin, potassium salt in 100 mM sodium citrate buffer solution, pH 5.0). AlphaImagerTM 1200 can be used for colonization of this microorganism Documentation and Analysis System (Flowgen, Lichfield, Staffordshire, UK) to observe that the reproduction of microorganisms will radiate bioluminescence over a period of time to produce an image of light emission. The brightness of light can be used as a thermal stability of fluorescein index. Then check the thermal stability of this microorganism, screen this microorganism on the basis of the brightness of the emitted light, and further use its characteristics to distinguish it. In some tests, five microorganisms will be cultivated at 42 ° C before detection. Hours, so thermally stable mutations can be singled out. Microorganisms isolated from the preliminary detection will be temporarily coated on the nylon film and grown overnight in LB media containing ayacillin. These coatings Will be sprayed with a fluorescent solution and observed in Alphaphalmager ™; advanced testing helps to clearly confirm the fluorescent activity of microorganisms in test tube analysis, and five microorganisms that may have thermostable enzymes will be analyzed in test tubes Thermal stability and fluorescence activity. The steps for performing fluorescence activity analysis in a test tube were performed at room temperature using a Promega luciferin analysis system (promega Corporation, madison, WI, USA.). The natural cell extract was added to 100 μl of Promega luciferin analysis solution (cocktail) (1 to 2 dilution) to start the fluorescence reaction, using Biotract M3 brightness The measuring instrument is used to measure the bioluminescent state produced by the results. 34 1239353 The preparation of natural cell extract is mentioned in the 101st issue of Promega Technical Journal. The whole number of five cW / cultured overnight is dissolved in the cell culture lysis reagent. The cell culture lysis reagents include 25 mM triphosphonate, pH 7.8, 2 mM dithiothreitol (DTT), and 2 mM 1,2-bisaminocyclohexane-N, N, N ', N'-tetraacetic acid ( 1,2-diaminocyclohexame-N, N, N ', N'-tetraacetic acid), 10% glycerol, 1% tritium X-100, 1.25mg / ml hen lysozyme, react at room temperature For 10 minutes, the natural lysate was then stored on ice before analysis. The nature of the enzyme is tested in a time-dependent deactivation study. Eppendorf tubes containing 50 μ1 of a complete natural cell extract are placed in water at a given temperature and the tubes are removed at a set time point and Before analysis, the samples were cooled with ice and the remaining fluorescence activity was expressed as a percentage of the original fluorescence activity. The relationship between the percentage of remaining activity and time is plotted on a logarithmic scale and used to calculate T1 / 21/2, T〃2 represents the time it takes to incubate at the specified temperature and lose 50% of the original activity of the enzyme. A logarithmic plot of percentage of activity versus time to determine the Tw2 天然 of the natural extract at 37 ° C (the time when the activity is reduced to 50% of the original activity). The cytoplasmic DNA in the five microorganisms with most of the above-mentioned thermostable luciferin will be sorted to determine the variation related to the thermostability of the enzyme. Cytoplasmic DNA can be prepared using the QIAGEN QIAprep Spin Miniprep Kit (QIAGEN Ltd, Crawley, W. Sussex, UK), followed by the method of the microcentrifuge operator 35 1239353 (QIAprep Miniprep Handbook 04/98). Through Babraham Technix, Cambridge, UK, according to the dideoxy chain termination method, an ABI PRISMtm 377 DNA Sequencer Ready Reaction Kit (Perkin Elmer Applied Biosystems) can be used to determine all DNA sequences. The result of this procedure can identify a new variant D357Y. The crystal structure of luciferin [E. Conti et al., Structure, 4 (1996) 287 ~ 298] shows that position 357 is located on the surface of the protein and is close to position 354, which can affect thermal stability and spectral properties. This indicates that This area is important for the thermal stability of the enzyme. D357Y is a unique thermostable mutation that turns into the most stable luciferin with an amino acid change. Example 2 Mutation of Unit Pointing To Create Other 357 Variations To evaluate different mutations at 357, site-directed mutations (mutagenesis) will be performed using Stratagene QuickChange ™ Site- Directed Mutagenesis Kit, (Stratagene, La Jolla, CA, USA), in which the cytoplasmic pPW601a J54, (PJW, MoD Report, 3/96) will be used in all unit-directed mutations. The products of all mutation reactions will be sent to the five slave // system XLl-Blue, [el4 '(mcrA') A (mcrCB-hsdsMR-mrr) 171 endAl supE44 thi ^ l gyrA96 relAl lacrecB red sbcC umuC :: Tn5 ( Kaif) uvrC [F9 36 1239353 proAB laclq ZnΛ15 (Tef) Amy Camr]] j Synthesis using Sigme-Genosys Ltd ·, Cambridge, UK, and using a smart implant system [AR Arkin et al ·, Biotechnology, ( 1992) 10, 297-300, W, Huang et al., Anal. Biochem. 218, 454-457] to design oligonucleotide primers to generate individual Primitives are more likely to produce variant families. In this method, amino acid substitutions of luciferin are made into a mutated genome. Oligonucleotide (and its complementary part) is used next:

Oligonucleotide Primer (5 '-> 3') amino acid substitution cacccgagggggat [tat] aaaccgggcgcgg (SEQ ID NO Y 4) cacccgagggggat [(gac) (tc) (c)] aaaccgggcgcggtcgg A, I, L ·, T, V, ( (SEQ ID NO 5) P cacccgagggggat [(t) (gat) (gc)] aaaccgggcgcggtcgg C, F, L, W, (SEQ ID NO 6) Y, X cacccgagggggat [(ac) (ga) (gc)] aaaccgggcgcggtcg R , S, K, N, g (SEQ ID NO 7) H, Q variant genomes can be pre-selected for thermal stability, and the number of tested microorganisms can be calculated using the formula [S. Climie et al ·, L Biol · Chem. 265 (1990) 18776-18779] N = [ln (lP)] / [ln ((nl) / n)] where N is the number of microorganisms detected, and n is a possible density at the target location. 37 1239353 The number of code units, and p is the probability of each password unit in the mixture being taken as a sample to be detected at least once. The calculation is performed according to P = 0.95. The mutation resulting from the unit orientation mutation will be analyzed. The activity and characteristics of luciferin were analyzed in a time-dependent thermal deactivation study. This method was used to determine that the expected mutation would grow to A260 «0.5 in 400 ml of LB media containing ayacillin, followed by addition of isopropyl β-thiogalactoside (IPTG) to a final concentration of ImM. In order to produce luciferin, the cells were cultured by shaking at 30 ° C for three hours, and then collected by centrifugation. The resulting cells were suspended in 10 ml of ImMDDT B-PER ™ protein extraction reagent. (Pierce Chemical Company, Rochford, USA ·) to generate a natural extract, followed by the B-PER ™ method for Maxi-Scale Bacterial Protein Extraction, 500 μl of the Sigma protein inhibitor solution (Product No. P8465, Simga, Saint Louis, Missouri, USA) was added to the B-PER ™ solution to suppress the internally produced protein. This cytolysin was then centrifuged at 30,000 grams for 30 minutes. Sulphuric acid is used to fractionate the natural extracts floating on the surface. The fraction of Shen Dian saturated between 30% and 55% contains fluorescent activity. This material will be suspended in 0.5 ml of Tris hydrogen chloride of ρΗ8 · 0, ImM DTT. , And used for thermal deactivation and spectrum research. Substituting D357L, T, V, W, R, I, S, K, N, and F, these changes will be resolved by the nature of the thermal deactivation of the natural extract in the test tube. The specially purified extract was diluted 1 to 11 and added to a hot 38 1239353 deactivated buffer solution: 50 ml of potassium phosphate buffer solution, ρΗ7.8, containing 10% saturated ammonium sulfate, ImM dithiothreotol and 0.2 % BSA 〇 Set a time period at 40 ° C or 45 ° C to incubate 11 μl of the complete protein solution, then cool with ice before analysis, and then use Promega luciferin reagent as described in Example 1 (1 to 2 dilution) to measure its fluorescent activity. The results are shown in Tables 2 and 3, and in Figure 1. The Tm 値 is determined by the natural extracts at 40 ° C (Table 2) and 45 ° C (Table 3). Table 3 Variation Tl / 2 D357W 2.5 D357F 6.5 D357Y 10.4 RWT < 1.0 Table 2 Variation Tl / 2 D357K 2.2 D357R 4.2 D357S 4.6 D357N 4.8 D357V 5.9 D357T 7.3 D357L 11.3 D357I 18.0 rWT < 1.0 Compared to the reorganized natural form All substitutions have been shown to improve thermal stability. Example 3 39 1239353 Changes in the wavelength of the emitted light Amino acid substitutions at position 357 can also be observed, which has an effect on the spectrum emitted by the enzyme in the test tube. A complete (250 μ1) microorganism, as described in Example 2 Grow overnight at 37 ° C, then spin in a microcentrifuge and remove the suspended solids. Cells expressing different variants of luciferin will be cultured in a citrate buffer solution (P5.0) containing 150 μ1 of D-luciferin, and the radiation emitted from the reaction of living organisms will be analyzed by measuring its emission spectrum. The light was measured using a SPECTRAmax® Microplate Spectrofluorometer (Molecular Devices Corp. California, USA ·). The mutations D357Y, F, and I (see Figure 2 (aHg)) can be observed from the spectral peaks as if the wavelengths changed greatly. These results are summarized in Table 4 below. In addition, the luminosity of variation in living organisms can be judged with the naked eye in a dark room. The variant of D357 shows a diverse color on the chromaticity spectrum, especially D357Y, F and I show on the emitted light. Obvious long wavelength shift. In some cases, such as D357F, the color of the incoming light changes appropriately. Not only is the wavelength λMAχ shifted, but also different contributions to the spectrum due to different visible light wavelengths. Recombinant natural (r-wt) enzymes will be used as a comparison with some of the 357 mutant light wavelengths λ MAX in living organisms. D357Y, F and I show an ideal shift in the maximum wavelength of the wavelength. 1239353 Table 4 Variation λ MAx (nm) rWT deviation (nm) rWT 558 D357K 556 -2 D357N 558 0 D357W 558 0 D357I 606 +48 D357F 611 +53 D357Y 613 +55 Example 4 Enzyme Characteristics with or without CoAJ # As described in Example 1, ammonium sulfate precipitation was used to partially purify D357Y. The partially purified D357Y enzyme (5 μ1) was mixed with 150 μ1 of Promega luciferin analysis reagent, and the other part was mixed with an equal amount of analysis buffer solution. In the buffer solution, there is no CoA (25 mM Tristricine, pH 7.8, 5.0 mM magnesium sulfate, 0.1 mM EDTA, 2 mM DTT, 470 μM D-luciferin and 530 μM ATP), and the radiation of the two reactions is measured. The spectra are plotted in Figures 9 and 10. In the absence and presence of CoA, the spectrum shows a marked difference in bioluminescence radiation, a significant shift λ MAX. In terms of the kinetics of the fluorescence response, the effect of CoA can also be observed by the difference in the RLU benchmark (relative light units, RLU stands for relative brightness unit). The difference in radiation increases the ability to use enzymes in the analysis of the presence of CoA. Example 5 Preparation of Double Variation and Its Special Features As described in Example 2, using the localized variation, in order to investigate any cumulative effects on thermal stability and the color of the emitted light, a double variant of E354I + D357Y was designed. The partially purified double variant, E354I + D357Y, was diluted 1 to 11 into a thermally deactivated buffer solution: 50 mM potassium phosphate buffer solution, pH 7.8, containing 10% saturated ammonium sulfate, ImM dithiothreitol, and 0.2 % BSA 〇 Incubate ΙΟΟΙΙ intact protein solution at 45 ° C for a period of time, then cool it with ice before analysis, and then use Premega fluorescence analysis reagent (1 to 2 dilution) to measure the fluorescence as before active. Compared with the individual single variants E354I and D357Y, the dual variants show a significant improvement in thermal stability (as shown in Figure 3). At a given Tm 値, it is 7.7 minutes. Deactivation at 45 ° C, thermal deactivation studies of partially purified double variants confirmed the increase in the thermal stability of the variants. It should be noted here that compared to the individual variants E354I and D357Y, the double variant exhibits a very deep red fluorescent light, which has an additive effect on the fluorescent heat. Both the recombined natural species and the natural extract of the double variant E354I + D357Y can be measured using the analysis buffer solution 42 1239353 mentioned in Example 3. In living organisms, the measured emission spectrum gives a λmax of 611 nm. In any case, the spectrum in the red light region has a greater contribution to the luminescence 'resulting in a deep red color to the naked eye; in The radiation spectrum of the natural extract shows a significant change in the shape of the spectrum, and its wavelength shift relative to rWT is 44nm (see Figure 4). In living organisms, the emission spectrum of the double mutant shows that the bandwidth of the wavelength peak (613nm) of the emitted light becomes sharp, and the contribution of the emitted light wavelength in the range of 540-560nm decreases. The significant effects of these mutations indicate the importance of bioluminescent light in this area of the enzyme. Example 6: Improved photon magnetism in living organisms. Compared to other variants at this position, the bioluminescence of the D357K variant and the cells is very bright. The flash kinetics of this enzyme can be measured with an illuminance. This meter can measure the rate of radiation of photons over time. The entire five Cb / human cell extract containing recombinant natural enzyme or variant D357 will be optimized into a fluorescent analysis solution. This solution does not contain any reagents that promote luminescence kinetics, such as auxiliary A. Measure The decay rate of photon emission to time (15 seconds), it can be seen that the variant D357K has a very obvious slowing effect (as shown in Figure 4). In other respects, the mutant enzyme has a reaction power. At least in the first 15 seconds of the reaction, it will be suppressed to a smaller distance than the recombinant natural enzyme. 43 1239353

Example 7 Combination of cassette-type mutation reaction steps for E354 and D357. Design of cytoplasmic pPW601aJ54 for one-cassette mutation reaction. In the cytoplasmic pPW601a / J54, two pairs of synthetic oligonucleotides were used to define two new unique definitions. The unit was introduced into the he gene (see below), all six stationary mutations were introduced into one of the genes, one Spel and one KρηI-defining unit, 63 reference pairs. The cytoplasm contains these new units called pPW601aJ54SpeI / KpnI. The existence and proximity of these defined units will make it possible to use combo-box identification to explore the amino acid positions 357 and 357 in the primary sequence of fluorescent heavy luciferin. Effects of random variation. Spel (a) 5? -GggctcactgagactacTAGTgctattatgattacacccg-3 'ntlQ21- nt! 060 (SEQ ID NO 8)

Spel (b) 5'-cgggtgtaacagaatagcACTAgtagtctcagtgagccc-3 (SEQ ID NO 9)

Kpnl (a) 5'-ggcgcggtcggtaaagtGgtAccattttttgaagcg-3 ntl078- ntlll3 (SEQ ID NO 10)

Kpnl (b) 5'-cgcttcaaaaaatggTacCactttaccgaccgcgcc-3? (SEQ ID NO 11) 44 1239353 In the recognition unit of nucleic acid bile, and those where the point mutation in the above case occurs, it is obvious that glycine is needed to generate this unit. The design of the second cassette (cassette) and a certain structure. When tempering (anneal) produces a double independent cassette, this cassette can be directly sutured into the cytoplasmic body pPW601aJ54SpeO / KpnI absorbed in the new limited unit, a pair of synthesis Oligonucleotide is synthesized, and this cassette is designed to introduce all possible combinations of the 20 naturally occurring amino acids in positions 354 and 357 of the primary sequence.

Looplib2A 5,-ctagtgctattctgattacacccNNG / CggggatNNG / Caaaccgggcgcggtcggt aaagtggta-3 ’(SEQ ID NO 12)

Looplib2B 5'- cactttaccgaccgcgcccggtttG / CNNatccccG / CNNgggtgtaatcagaatag ca-3 '(SEQ ID NO 13) Take 2 μ2 of oligonucleotide from each loop gene segment, and a buffer containing 50mM Tris hydrogen chloride, pH7.4, 25mM sodium chloride The solution was mixed and heated at 100 ° C for 3 minutes. The solution was then slowly cooled in a hot block of less than 50 ° C to restore the complete sequence. Tempered oligonucleotide was then sutured to the cytoplasm pPW601aJ54SpeI In / KpnI, this cytoplasm has been absorbed by Spel and κρηΙ. Electroporation followed by the entire suture reaction was used to transform the five HB101 cells. After 45 1239353 electroporation, the transformed cells were coated on LB agar plates containing 50 pg / ml ampicillin and grown overnight at 37 ° C. The following day 869 multiplying microorganisms will be randomly picked from the board and used to inoculate 1 ml of a 96 square plate (Beckman) containing LB of ampicillin, the board is covered and the cells are at 37 ° C. Shake to grow all night. Step 3 Detect randomly sampled microorganisms in living organisms. In the next morning, transfer 50 μ1 of the whole body cultured in a fixed state overnight to two clean plastic circular plates (Dynex), whose bottom well is 96 micrometers. For fatty acid fixation (microtitre), one plate is covered and incubated on a heated block for 8 minutes (the surface temperature of the block is 45 ° C), while the other is maintained at 37 ° C. Next, the fluorescent activity in the cells taken from the two plates was measured and recorded in a living organism. The detection was performed at room temperature. By using pH 5.0, 50 μ1 containing 0.5 mM D-luciferin had 100 mM sodium citrate buffer was added to the well, and the plate was then transferred to an image capture system (Alpha Imager). The light emitted by heating and controlling the microbiota was accumulated for 1 or 2 minutes, and the image was recorded. On the thermal film. Seventy-nine microbiota exhibiting the best bioluminescence were selected in the second round of testing. The best luminosity was determined by the brightness of the image recorded on the membrane. This time, these microbiota will be placed before analysis. Incubate on heating block for 16 minutes. Of the 55 asexually reproduced individuals selected by the thermostability test in living organisms, 25 will be selected for spectral analysis of live organisms. 46 1239353 Analysis of these asexually reproduced individuals in LB at 37 ° C Grow overnight and centrifuge at 200μ1 in the next morning. Then place five cells and nine in a white plastic microlipid fixation plate and use a Molecular Devices Spectramax 96 well plate Huorimeter to analyze living organisms. The emission spectrum of each mutated luciferin is shown in Table 1 ^ above. Step 4: Confirm the mutation. Sort the microorganisms using the gene-specific sorting primitive 値, and use the detection of 25 microorganisms selected in the living organism to prepare the cytoplasmic DNA. The mutation results in the amino acids with positions 354 and 357 changed in the initial sequencing. It can be confirmed that one of these mutations also contains an additional mutation result of an amino acid substitution at position 1351, see Table 5. 1239353 Table 5 Peaks (nm) of the wavelengths of the mutation enzymes 1 E354V / D357Y 614 2 E354I / D357Y 612 3 E354C / D357Y 612 4 E354R / D357Y 600 5 E354S / D357Y 612 6 E354N / D357Y 608 7 E354K / D357M 556, 606 8 E354R / D357L 588 9 E354W / D357W 610 10 E354H / D357W 606 11 E354R / D357F 596 12 E354K / D357F 608 13 E354S / D357F 610 14 E354M / D357F 610 15 E354A / D357R 556 16 E354A / D357F 610 17 E354T / D 18 E354A / D357N 560 19 I351M / E354R / D357V 606 20 E354S / D357V 556, 608 21 E354R / D357W 600 22 E354R / D357M 596 23 E354R / D357S 592 24 E354N / D357S 600 rWT E354 / D357 5yi 48 1239353 where rWT means reorganization Natural form. A series of mutated luciferin was selected from the analysis of thermal stability in the living organism. 'Most luciferin also has a large change in the emission spectrum of the living organism'. Many are in the long wavelength of light (& gt (580nm); some spectra also show a significant narrowing of the bandwidth of a single peak at 610-614nm. A water-repellent substitution of E354 and D357, and the use of an aromatic amino acid 'like E354V and D357Y, respectively, will have the largest change in the spectrum of living organisms, and a single band with a narrow bandwidth will appear at about 612 nm. Absorption peak. Example 8-Examination of a tube in a test tube [[Thermal stabilizer uses lysis to make cell extracts of selected microorganisms, and in a thermal deactivation experiment determines the thermal stability of the camphorin of each extract, 50 μ1 Each extract was placed in an eppendorf test tube and incubated at 45 ° C for 4, 9 and 16 minutes. Remove the entire φ 'at the appropriate time point and measure its remaining activity. Table 6 shows all the variations. Enzyme vs. recombinant natural species, the percentage of remaining activity versus time. 49 1239353 Table 6 Enzyme number remaining activity percentage after incubation at 45 ° C (see Table 5) 0 minutes 4 minutes 9 minutes 16 minutes 1 100 95 87 75.4 2 100 99 84.7 67.7 3 100 82 73 53.3 4 100 84 89 71.4 5 100 85 72.2 53 6 100 93 84.8 71 7 100 63.7 31 11.7 8 100 58.6 19 4.9 9 100 85.4 65.3 42.3 10 100 65.5 27.8 10.6 11 100 88.6 70 54 12 100 90 69 52 13 100 83 60.5 39 14 100 80 61 39 15 100 1.7 0.1 nd 16 100 90 76 63 17 100 91 78 60 18 100 19 1.8 nd 19 100 17 1.4 nd 20 100 17 1.1 nd 21 100 71 63 34 22 100 80 40 21 23 100 29 4 0.6 24 100 28 4 0.4 25 (D357K) 100 0.1 nd nd rWT 100 0.05 nd nd where nd means not done. 50 1239353 The results show that most of the thermally stable luciferins are those with aromatic amino acids at position 357 (Υ, F or W) and a large water-repellent amino acid (V or I) at position 354, polarity Amino acid (N) or positive charge (K or R). Example IX. The effect of growth maggots on the radiation spectrum of living organisms. The radiation spectrum of ugly a // BL21 (DE3) cells showing mutated luciferin D357Y or E354K + D357M (7 in Table 5), The effects of different carbon sources will be analyzed and detected. The 50 ml cell population was cultured on the central log phase of the LB medium, and then collected by a centrifugal separator. The cells were resuspended in 1 ml of sterile distilled water, and 100 μ1 of the suspension was used for To inoculate 5 ml of fresh LB in 25 ml Sterilin tubes, M9 minimum medium + 2mM sodium citrate, or M9 minimum medium + 2mM glucose, these populations will continue at 37 ° C with shaking Grow. After 90 minutes (D357Y) or 120 minutes (enzyme 7), 200 μ1 of the cell culture is centrifuged and resuspended in a 150 μ1 buffer solution. This buffer solution is ρΗ5.0 · 0 and contains 0.5 mM D. -Luciferin 100 mM sodium citrate buffer. The resuspended cells were then placed in a microfatty acid fixation plate and the Molecular Luminescence Spectramax 96 well plate fluorimeter was used to analyze the bioluminescence emission spectrum of each variant luciferin emission in a living organism. Figure 7 and Figure 8 show. The results show that from a rich medium (LB) (Figures 7a, 8a) to a specific medium with acetate (Figures 7b, 8b) or glucose (Figures 7c, 8c) as the minimum carbon source, it emits light. The shift to longer wavelengths and the effect of shorter wavelengths is reduced. Example + Purification of Recombinant Natural Species and Mutant Fluorescein and Their Fluorescence Characteristics To analyze the effect of Cofactor CoA on the spectrum of the bioluminescence response, the recombinant natural enzymes and mutant Fluorescein D357Y and E354I + D357Y will Purified into the same species, combined with a 143 amino acid carbohydrate bond model (CBM), these three luciferins can be purified from anaerobic bacteria / Vrwyca. This CBM is mentioned in the selective attachment of acid to the expanded cell membrane And soluble carbohydrates, galactomannan and glucomannan, will form a simple single-step homogeneous purification benchmark. The fluorescein fused to CBM can be bonded to the cell membrane in the natural cell extract, washed with a solvent, and then washed with soluble polysaccharides. The fusion protein used in this method will be used in In the analysis, to measure the wavelength of light emitted in reactions containing different amounts of co-A, enzyme (5μ1) will be added to 100μΙ of the analytical reagent, which is 25mM Tris-tricine, pH7.8, 5.0mM Magnesium sulfate, 0.1 M EDTA, 530 μM ATP and 470 μM D-luciferin, which contained different amounts of co-A. Figures 9 to 11 show the effect of increasing the concentration of auxiliary A on the radiation spectrum of light emitted by purified fluorescein D357 and E354I + D357Y. Analysis of the bioluminescence emission spectrum of five cells from five cells in a living organism. 52 1239353 The light spectrum contains cells containing the C-terminus of firefly luciferin fused to the mold's CBM. This analysis did not show similarity to natural luciferin There are no obvious differences in the cells, and the spectrum of bioluminescence emitted from a commercial source of purified recombinant luciferin (Promega) was analyzed in a test tube similar to that of a fusion protein. Therefore, it can be judged that the significant difference is related to the concentration of CoA. When the concentration of auxiliary A increases, the spectral contribution will change, and at the highest CoA concentration, the spectrum will be a significant absorption peak at 610nm from 590-630nm. Affected by this, the double mutation has the most obvious effect on this spectral shift, in which there is a single single absorption peak with a narrow bandwidth at a wavelength of 610nm (see Figure 11). Example + — Production of a synthetic 7¾plus // 7 valence-like luciferin with a mutation at 214C / 354K / 357F Using the synthesis technique mentioned in the above description, a synthetic luc gene was designed and combined from an oligonucleotide pair This gene sequence was designed to form a luciferin with amino acids 214C, 254K and 357F. 29 pairs of overlapping synthetic oligonucleotides were synthesized by Sigma-Genosys Ltd, purified by PAGE, and sutured at three junctions (IDRIS 1, 2 & 3, see Figure 13) at approximately 550bp, followed by joining each Each was connected to the vector pBSK (+), and the final structure was used to change the ugly XLl-Blue cells. -Asexually propagated individuals containing conjugated inserts to prepare cytoplasmic DNA and sequenced to identify ORF fireflies. The presence of (n-1) oligonucleotides (byproducts of oligosynthesis) at the junction will complicate the architecture. Confirmation 53 1239353 The only correct DNA sequence that joins IDRIS 2 and PCR will be used to confirm one of IDRIS3 Conjugation, in which IDRIS3 has a base pair deleted at the 5 'end of the architecture, and conjugation of the complete ORF is accomplished by joining a mixture of cytoplasmic bodies containing IDRIS1 and IDRIS2 and 3. Next, the conjugated DNA was used to alter the eight cW XLl-Blue cells, and the asexually reproduced individuals containing active enzymes were selected by analysis in a living organism. Several asexually reproduced individuals were selected and sorted to determine, for example, The presence and accuracy of the sequenced synthetic luc genes shown in Figure 14, the complete ORF is called IDRIS (FA). The synthetic gene is inoculated into the vector pBSK (+) located between the BamH I and Sal I in the polylinker. In this position, the gene will not be in the architecture with alpha shoulder, and it has the same function as the lacy promoter. A clear distance. In any case, it will produce enough luciferin so that it can show the basic characteristics of enzymes. Promega lysis method is used to prepare natural cells containing IDRIS (FA) pentagram // Xll-Blue cells from microorganisms cultured overnight. Extract. Next, the thermal stability of the enzyme in the extract was tested at 50 ° C for more than 20 minutes, and compared with the thermally stable variant E354I + D357Y, compared with the variant E354I + D357Y, a new cryptographic unit of three variants was used Obviously has better thermal stability (see Figure 15).

Claims (1)

1239353 Date of amendment: June 6, 1994 is No. 89122956 Chinese Patent Scope Unlined Revised ff, '-ii "Application scope of patent: 1. A recombinant protein with luciferase activity and natural luciferase At least 90% identity, where the natural fluorescent enzyme is selected from Photinus pyralis, Luciola mingralica, Luciola cruciata or Luciola lateralis, Hot aria parvula, Pyrophorus plagiophthalamus 'Lampyris noctiluca' Pyrocoelia miyako J compared to natural luciferase The amino acid fragment of the 357 fragment corresponding to M AK / ^ valent fluorescent enzyme is mutated, and this fluorescent enzyme can emit light of different wavelengths than natural fluorescent enzymes, and / or can be compared with natural fluorescent enzymes. Increased thermal stability. 2. The recombinant protein described in item 1 of the scope of the patent application, wherein the natural fluorescent enzyme sequence is an enzyme sequence taken from Photinus pyrahs, LucioL. Mingrahca, oracyaG, or L / ycio / a enzymes [J. 3. The recombinant protein according to item 1 of the scope of patent application, wherein the natural fluorescent enzyme sequence is selected from the group consisting of μ, LWo / s mingralica, Luciola cruciata M Luciola lateralis, Hot aria parvula 'Pyrophorus plagiophthalamus' Lampyris noctiluca, or The sequence of several fluorescent enzymes, and the fragment 357 corresponding to the fluorescent enzyme is an amino acid fragment other than aspartic acid or glutamic acid. 4. The recombinant protein according to item 1 of the scope of the patent application, wherein the fragment 357 corresponding to the fluorescent enzyme is an amino acid fragment other than aspartic acid, bonded amino acid or valeric acid. 5. The recombinant protein described in item 1 of the above patent application range, wherein 55 1239353 corresponding to the P-fluorescent enzyme fragment 357 is an uncharged polar amino acid. 6. The recombinant protein according to item 5 of the scope of the patent application, wherein the fragment 357 corresponding to ακμ / λ? Fluorescent enzyme is tyrosine, phenylalanine or tryptophan. 7. The recombinant protein according to item 6 of the scope of the patent application, wherein the fragment 357 corresponding to the corpse py / Ws fluorescent enzyme is tyrosine. 8. The recombinant protein as described in item 1 of the above patent application scope, which includes at least one of the following variations compared to natural fluorescent enzymes: (a) the amino acid 354 corresponding to the fluorescent enzyme (in hew △ There is a variation in the amino acid fragment of 355 in fluorescent enzymes; (b) the amino acid fragment corresponding to position 215 of the external / 7 AXπ-like fluorescent enzyme (217 in Z⑽Ws fluorescent enzymes) is a different type of taboo Hydrophobic amino acid; or (c) an amino acid fragment corresponding to fragment 214 of the luciferase of the outer plate, or a fragment 216 corresponding to Luciola mingrelica, Luciola cruciata, or Luciola lateralis; ⑷ corresponding to pyrWs fluorescent Photoenzyme fragment 232, or an amino acid fragment corresponding to the 234 amino acid fragment of Luciola mingrelica, Luciola cruciata, or Luciola lateralis luciferase; (e) Amino acids corresponding to fragment 295 such as μ pyrWs luciferase, M series should be 、 Amino acid fragment of 297 amino acids in Luciola mingrdica, Luciola cruciataM Luciola fluorescent enzyme fragment; ⑴ corresponding to amino acid 14, pyrWs fluorescent enzyme, Or corresponding to 16 1239353 to Zwc / oh plate fragment 16, or amino acid fragment corresponding to / c / 7 / d · Hor or / Wera valence fragment 17; (g) corresponding to all α such as μ ακμ valent enzyme Amino acid 35, or a fragment 37 corresponding to Qiu / Άα, or an amino acid fragment corresponding to fragment 38 of hcioh cn / c or h or / WerWs; ⑻ an amino acid fragment 105 corresponding to yang plus luciferase, or The amino acid fragment corresponding to the fragment 106 of hcio / a mi and rAh, or the fragment 107 corresponding to hcio and 107, or the fragment 108 of the hci0 / a valence gene; ⑴ the amino acid fragment 234 corresponding to the pyra / k luciferase, Or ying ying, wheat \ Luciola mingrelica, Luciola cruciata or Luciola valence fragment 236 amino acid fragment; (J) amino acid fragment 420 corresponding to Hori, such as μ pyrWs luciferase, or ying ying, 3 \ Luciola mingrelica, Amino acid fragment of Luciola cruciata or Luciola valence fragment 422; (k) Amino acid fragment corresponding to amino acid fragment 310 of the luciferase, or MMM Luciola mingrelica, Luciola cruciata or Luciola fragment 312; appears in the sequence with the natural species Corresponding to the amino acid fragment is different, but also compared to the naturally occurring amino acids having a fluorescent enzyme at these positions, these fluorescence enhancing thermal stability of enzymes. 9. The recombinant protein according to any one of the above patent claims, wherein the amino acid 354 (or 356 in the case of luciferin) corresponding to the γ pyra / k luciferase is mutated. 1239353 10. The recombinant protein as described in item 9 of the scope of the patent application, wherein the amino acid fragment corresponding to fragment 214 such as fluorescent enzyme or the fragment 216 corresponding to mingrelica, Luciola cruciata or Luciola lateralis Mutates into a different water-repellent amino acid. 11. A nucleic acid, wherein a fluorescent enzyme is encoded according to any one of the scope of the aforementioned patent applications. 12. The nucleic acid according to item 11 of the patent application scope, which includes a synthetic gene. 13. A nucleic acid as described in claim 11 in the scope of the patent application, wherein the use of a cryptographic unit is applied to a single dominant host and / or a unique restricted location is introduced. 14. The nucleic acid according to item 11 or item 12 of the scope of the patent application, which includes the nucleotide 9-1661 of SEQ ID N01, or a sequence at least 90% identical thereto. 15 · —A kind of biochemical fluorescence test group, including the protein described in the first patent application scope. 16. The biochemical fluorescence test group according to item 15 of the scope of patent application, which further includes luciferin. 17. An analysis method used to determine the presence of coA in a sample, wherein the analysis includes adding a fluorescent enzyme as described in item 1 of the scope of the patent application to a sample suspected of containing CoA, and Other reagents required for the luciferase / luciferin reaction: measure the wavelength of light emitted by the sample; and correlate the result with the presence or absence of CqA. 18. The analysis method described in item 17 of the scope of patent application, which is used for the diagnosis of diabetes.