TWI239353B - Thermostable luciferase mutant enzymes, nucleic acids encoding them and the use thereof - Google Patents
Thermostable luciferase mutant enzymes, nucleic acids encoding them and the use thereof Download PDFInfo
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1239353 九、發明說明: 本發明是有關於一種新酵素,且特別是有關於一種變異 螢光素酵素,相較於天然酵素具有特殊性質,對於DNA編 碼蛋白質,可以用這些酵素於分析上,也有關於含有這些 酵素的測試工具。 螢火蟲的螢光素會催化存在ATP、Mg2+以及氧分子中的 螢光素的氧化,而產生光,此反應的量子產率約爲0.88, 發光的特性以使其廣泛的應用在光度分析上,其中ATP的 量可以量測出來,包括酵素的分析例子可見於歐洲專利 EP-B-680515與WO 96/02665,但是有其他例子是在實驗室 中常用到的。 螢光素可以由昆蟲體內直接取得,特別是甲蟲類像是螢 火蟲(firefly or glow-worm),可以取得螢光素的特別物種包 括曰本的 GENJI 或 KEIKE 螢火蟲(fireHies)、 cruciata 與 Lucioh !atera!is、東歡的營火蟲i(i]jef\y) Luciola 、北美的螢火蟲(firefly) PAot/nus pyralfs 與螢火蟲 (glow-worm) Lampyris noctiluca. 無論如何,因爲在編成這些酵素的很多基因中已經被複 製及排序出來,所以這些酵素也可以用重組DNA技術排列 出來。重組的DNA排列編碼的酵素可以用來使微生物,像 是万· coli產生變異,使其產生預期的酵素產品。 當在實驗室進行分析時,這些酵素放射出來的光都很相 近,假如其波長可以改變,將會有使特殊檢測器的辨讀更 容易,或是有助於使用在需要多重紀錄器的系統上,比如 1239353 用於在同樣的樣品中監測不同的事件。辨識紀錄分子的一 個方法爲使用在特定波長放射光的螢光分子,這方法可使 用自甲蟲或螢火蟲中不同的種類得到之含有螢光素的紀錄 分子來達成,一種可行的方法就是利用重組DNA技術來製 造變異的螢光素,藉以在訊號波長上產生變化,此類變異 的例子可見於專利W0 95/18853。 此外,天然與重組型態的螢光素的熱穩定性爲,當暴露 在高於30°C的溫度時,特別是高於35°C時,螢光素會很快 的喪失活性,這種不穩定在高溫環境下使用與儲存時會有 問題產生,或是假如分析必須在高溫反應條件下進行,像 是爲了增加反應速度,也會有問題產生。 由專利EP-A-524448與W0 95/25798中可以知道變異的 螢光素有增進的熱穩定性,在這些敘述中首先提到在曰本 螢火蟲螢光素中的位置217上有一個變異的變種螢光素, 特別是利用一個異白氨酸(isoleucibe)片段來取代羥丁氨酸 (threonine)片段,而後提至ίί變種蛋光素與自仰如·/^ pyralis、Luciola mingralica ' Luciola cruciata 或 Luciola 取得之螢光素相比,具有超過60%的相似性,但其 中對應於价的片段354或種類的356 片段之氨基酸片段有變異成與麩氨酸鹽(glutamate)相異,且 特別與麩氨酸鹽、天門冬酸鹽(aspartate)、脯氣酸(proline) 或氣基乙酸(glycine)相異。 共同申請的英國專利案號9823468.5的申請案,以及源 自於此申請案的國際專利申請案提到更多這類的變異,在 1239353 其中,提到蛋白質,其中具有螢光活性,且與天然螢光素, Photinus pyralis ' Luciola mingralica ' Luciola cruciata 或/WWZ?酵素,至少有60%的相似性,但是其中 在蛋白質的各種位置上有變異,在這之間包括 (3〇街應PAoijfnus pyraiis的螢光素之214片段以及LucioJa mingralica ' Luciola cruciata 或 Luciola lateralis 營光素 Τί6 片段的氨基酸片段;或是 (b) 辑應於Photinus pyraiis的螢光素之232片段以及LucioJa mingralica ^ Luciola cruciata WL Luciola lateralis 234 片段的氨基酸片段;或是 (c) 繫應於PJiotinus pyrahs的螢光素之295忤段以及LucioJa mingralica ' Luciola cruciata ^ Luciola lateralis 297 片段的氨基酸片段。 這些申請案發現透過在螢光素的不同位置上改變(或加 入)氨基酸,可以使放射光的波長有很大的位移,並且/或可 以改進酵素的熱穩定性。此外,可以改進放射光的光子磁 力(proton flux),使得酵素更適用於在活的有機體(//? κ/κο) 避免哈光動力的分析,或是適用於在沒有CoA或其他’發光 動力感應’的化合物的試管中(“ Ρ7Υ/Ό)。 本發明提供一種重組蛋白質,具有螢光活性,且與天然 螢光素至少有60%相似性,其依序組成酵素,其中相較於 樹應的天然螢光素,澍應於PAotimis PyrWs螢光素的357 片對的氨基酸片段有變異,這樣螢光酵素與天然螢光素相 比可以放出不同波長的光,且/或相較於天然的螢光素可以 1239353 增加熱穩定性。 天然的螢光素序列可以作爲本發明重組型態的主要成 分,其中包括 Photinus pyralis、Luciola mingralica、Luciola cruciata ^ Luciola lateralis ' Hot aria parvula、Pyrophorus plagiophthalamus Lampyris noctiluca ' Pyrocoelia mivako 、 Photuris pennsylvanica 或 PAr/zc^Ar/Xrailroad-worm —見 Biochem. 38 (1999) 8271-8279)。由物種取得的生物發光酵素 可以使 用基質 D- 螢光素 (4,5-dihydro-2-[6-hydroxy-2-benzothiazolyl]-4-thiazol carboxylic acid)來放出光,五生物發光酵素可以作爲本發明 變種酵素的主要成分。 可以作爲本發明之重組型態的主要成分之特定的天然 營光素序列包括 Photinus pyralis、Luciola mingralica、Luciola cruciata Luciola lateralis ' Hotaria parvula、Pyrophorus plagiophthalamus Lampyris noctiluca ' Pyrocoelia mivako ' Ϊ^Χ 及 Photuris pennsylvanica ° 特別的是,螢光素就是可以由 mingralica、Luciola cruciata 或 Luciola lateralis 傳素中取魯 的廢素。在 Luciola mingralica、Luciola cruciata 或 Luciola 酵素中,適當的氨基酸片段是位在序列中的位置 359 上。 所有各種螢光素的序列顯示這些螢光素在其相似性之 間保存有相當重要的分界,這表示透過序列的檢查來偵測 大部分相似的區域,可以很容易的決定在這些酵素序列中 1239353 相對應的區域,雖然在某些條件下,爲了決定對應的區域 或是在各種序列之間的特定氨基酸,必須用到商用軟體(像 是由 University of Wisconsin Genetics Computer Group 提供 的” Bestfit” ;請見 Devereux 等人(1984)之 Nucleic Acid Research 12·· 387-395)。選擇或另外,可以利用參考L. Ye等 人之 BioChim· Biophys Acta 1339 (1997)39-52 來決定對應的 氨基酸,其著作中顯示出酵素序列、包括編號,其編號系 統將與本應用作聯結。 有關於與的螢光素中的片段357相對應 的氨基酸片段可能的變化,大部分天然序列在這個位置會 有一個氨基酸片段(丁氨二酸(aspartic acid)或是麩氨酸 (glutamic acid)),其例夕f 是在 的螢光 素的某些型態上,在其中對應片段(355)爲非極性片段的纈 草胺酸(valine),或是在螢光素的某些型態中其 對應位置爲在Pvc}r或在PhRE中的V354,是L354的白氨酸, 因此通常用在這個位置作爲取代氨基酸爲丁二氨酸、麩氨 酸、纈草胺酸或白氨酸以外的氨基酸。 因此,在大部分的情況中,氨基酸片段是用沒有酸性的 片段來取代,包括鹼性的氨基酸像是離氨酸(lysine)或精氨 酸(argimne),非極性的氨基酸像是白氨酸、纈草胺酸或異 白氨酸,沒有帶電的極性氨基酸像是酪氨酸(tyrosine)、天 冬素(asparagine)、麩氨酸、苯基丙胺酸、絲氨基酸(serine)、 色氨基酸(tryptophan)或羥丁氨酸。且特別是用沒有帶電的 極性氨基酸,像是酪氨酸、天冬素、絲氨基酸或羥丁氨酸 1239353 來取代,其中在這個位置用於取代的較佳氨基酸片段爲酪 氨酸、苯基丙胺酸或色氨基酸,且最佳的是酪氨酸。一搬 來說,芳香族的片段在這個位置可以增加最大的位移,且 可以有助於熱穩定性。 天然的序列包括在這個位置的非酸性氨基酸片段,可以 變異成不同的非酸性片段。 利用此方法改變酵素可以發現,螢光素放射的光波長被 改變,在一些情況中波長可以往光譜的紅光端位移50nm。 因此,D357Y變異价的螢光素放射的光波長 有一部份在612nm,相較於天然的酵素,其放射光的波長 爲 562nm。 50nm的波長位移在分析應用上具有不可忽視的影響, 當此位移的量可以輕易地在光譜上定義出來。不同顏色的 螢光素在基因表現硏究中可以用來作爲標示分子,可以同 時監控多於一個基因,比如如W0 95/18853中的說明,多 重分析測試的進行也可以用螢光素作爲標記。 事實上,在此情況中的光顏色爲深紅色,此顏色在分析 方法學中特別有用,紅色的變異在分析一個含有色素或其 他會吸收叫短波長的光之化合物的ATP溶液時會很有用, 舉例來說,紅色的溶液不會吸收紅光,紅色溶液,時常用 於這樣的分析的對象的例子包括血液樣品,或是真核細胞 (eukaryotic cell)培養媒介的溶液,其中可能會含有一種紅色 的pH指示劑。 當使用比色(colourimetric)試劑像是螢光素的一種混合 11 1239353 物時,產生深紅訊號的能力是很有用的,特別是在樣品中 的另一個試劑產生一個綠色訊號時,在光陰極光譜分析中 使用的光電倍增管可以被設定用來偵測一或兩個在訊號樣 品中產生的峰(peak),換句話說,可以在同一個樣品中,區 分出由紅與綠放射子產生的光子磁力。 此外,可以發現波長位移可以透過輔因子(cofactor)的輔 膊A(C〇A)的存在來加以影響,此特徵提高了將此酵素應用 在輔因子的分析的可能性。 如下所述,輔因子的輔膊A在試管中對放射光譜的影 響被謹慎的審查過,輔晦A的濃度增加了光譜分布的改 變,而且在CoA的最高濃度下,透過在590-630nm區域之 間,在610nm有一個標示峰的波長可以控制光譜。 因此,根據本發明的另一方面,提供一種分析法,用以 在樣品中偵測CoA的存在,其中分析法包括加入一個可能 含有CoA的樣品,包括上述的螢光劑以及用來產生螢光 (luciferase/lucufenm)反應所需要的其他試劑;測量由樣品放 射出來的光的波長;將結果與CoA的存在與否作關聯。 在成長狀態或活性的細胞的偵測中這樣的分析會很有 用,比如在微生物或真核細胞。 舉例來說,在K 細胞中的CoA的濃度相當高,且 對新陳代謝有好的作用,本發明的變種酵素可以用來監測 有機體新陳代謝的狀態,特別是在活體中的CoA的濃度, 因爲放射光波長的變化與CoA的濃度有關。當在製造抗生 素(比如在鏈黴菌屬(streptomycetes))時,CoA爲重要的主要 12 1239353 代謝物,在此情況下上述的分析法會特別有用;細胞中的 CoA濃度也是脂肪酸生物合成的一種重要的指示劑,且在 細胞處於飢餓狀態會有變化,一些新陳代謝失調’像是癌 的生成(carcinogenesis)與糖尿病,會在脂肪酸的代謝上出現 異常,而導致CoA有不正常的量,本發明的分析可以用在 診斷這些情形上。舉例來說,在一個細胞樣品,比如是來 自於病患的血液樣品中,可以在分析中,透過測量本發明 的一種螢光素放射出來的光波長來決定CoA的量,此結果 可以與一個由健康細胞取得之樣品作比對,以決定是否有 波長的改變,而因此存在一個修正過的CoA的量,這可以 用來顯示病患的病症狀況。在分析之前,會用一種已知的 細胞溶解藥劑先將細胞加以破壞溶解。一般認爲在位置357 的氨基酸片段必須與輔晦A的接合點聯結,當螢光酵素的 表面利用解析度爲1埃(angstrom)被描繪出來時(使用Tripos Ltd·出品的SYBL蛋白質模型軟體),一個小的極性開口會 被標示出來,此開口被劃線標示爲片段H310、E354與 D357,且被測量出在8-10埃之間。由分子的上方來看,此 開口就像是一個較大開口的一部份,被劃線標示爲H310、 E354、D357與1232,片段H310與E354係用以作爲一個橫 跨過兩個較小的開口外觀的裂縫的一座橋(如第8圖所示)。 不需要用原理來連接,當酵素放在溶液中以提供一個較 大的開口允許CoA連接時(約12埃的深度以及約8埃的寬 度),橋接的片段可能具有足夠的彈性來脫離,這與能量計 算的結果相符合。 13 1239353 當表現本發明的螢火蟲螢光素變異的五c〇/y細胞在不 同的碳原料上成長時,可以觀察到在活體中的放射光光譜 改變;以醋酸鹽或葡萄糖作爲爲一的碳原料,由一個富含 觸媒(LB)到一個定義最低限度的觸媒的變化結果會使放射 光往較長的波長位移,而降低較短波長的貢獻量,這或許 可以在分析上作爲控制放射光的波長的進一步的方法。 在蛋白質的位置357知變異已被發現會造成熱穩定的 提昇。 在蛋白質的螢光素活性沒有過度的妥協時,可以提供含 有進一步變異的蛋白質,這些變異適合增進酵素的特性, 或是使其較適用於某些方法中的某些目的,這可能表示這 些變異的結果增進了熱穩定性及/或顏色的位移特性,以及/ 或酵素的ATP的Km。增進顏色位移的變異的例子可見於 W〇95/18853的專利中;影響Km値的變異的例子則在W〇 96/22376的專利中以及國際專利申請案號爲 PCT/GB98/01026的申請案中提到。 一般來說,變異的影響已被發現被附加在特性變化的項 目上。 本發明提供之變異螢光素可以包括其他特殊的變異,相 較於天然的螢光素,這些變異可以增進熱穩定,特別的是 至少包括一種下列提到的情況: (a)對應於螢光素的氨基酸354(在螢 光素中則是356)的氨基酸片段有變異; 0>)繫應於PJiodmis 營光素位置215(在LucioJa營光 1239353 素中則是217)的氨基酸片段是一種不同的忌水的 (hydrophobic)氨基酸;或者是 (c) 對應於登光素的片段214,或對應於 Luciola mingrelica ' Luciola cruciata ^ Luciola lateralis 素的片段216的氨基酸片段; (d) 對應於价螢光素的片段232,或對應於 Luciola mingrelica ' Luciola cruciata M Luciola lateralis 素的片段234氨基酸的氨基酸片段; (e) 對應於螢光素的片段295的氨基酸,或 繫應、於 Luciola mingrelica、Luciola cruciata或 Luciola lateralis 螢光素的片段297氨基酸的氨基酸片段; (f) 對應於/¾螢光素的氨基酸14、或對應到 LucioJa mingrehca 的汽段 16、或截應到、LucioJa cruoiata 或 的片段17的氨基酸片段; (g) 對應於用^/7似螢光素的氨基酸35、或對應到 Luc】’oL· mingrehca 的肖段 3Ί、或絮應肩、Lucioh cruciata 或 的片段38的氨基酸片段; (h) 對應於价螢光素的氨基酸片段1〇5、或對應 到 Z^C/o/3 的片段 106、或對應到 約 的片段107、或是基因的片段108的氨基酸 片段; ⑴對應於螢光素的氨基酸片段234、或對應 兔 Luciola mingrelica、Luciola cruciata 東 Luciola lateralis 饱 片段236的氨基酸片段; 15 1239353 (j) 對應於螢光素的氛基酸片段420、或對應 3\ Luciola mingrelica ' Luciola cruciata M Luciola lateralis 6¾ 片段422的氨基酸片段; (k) 對應於螢光素的氨基酸片段310、或對應 3\ Luciola mingrelica ' Luciola cruciata M Luciola lateralis 片段312的氨基酸片段; 在與天然種類的序列上出現的氨基酸相對應的片段中是不 同的,而且相較於在這些位置上具有天然螢光素的氨基 酸,上述這些螢光酵素具有增進的熱穩定性。 因此,本發明提到的蛋白質的較佳實施例是變異的天然 螢光素,其中有超過一個以上的氨基酸與在適當的天然酵 素中相對位置的氨基酸是不相同的,其中的氨基酸數量比 如達到100個氨基酸片段,較佳是不會超過40個氨基酸, 且最好是達到30個氨基酸。 因此,在一個較佳實施例中,本發明的蛋白質包括 螢光素,其中除了上述的357位置上有變異 以外,還至少包括下列情況之一: a) 對應於P力螢光素的氨基酸354爲除了麩氛酸 鹽以外之氨基酸片段; b) 對應於py/Ws螢光素中的位置215之氨基酸片段 爲一種除了丙氨酸以外的忌水的氨基酸; c) 對應於螢光素中的位置214爲經丁氨酸以 外之氨基酸片段; d) 對應於螢光素中的位置232爲異白氨酸之 1239353 氨基酸片段; e) 對應於螢光素中的位置295爲苯基丙胺酸 以外之氨基酸片段; f) 對應於Λ^/^似 pyrWs螢光素中的氨基酸14爲苯基丙胺 酸以外之氨基酸片段; g) 對應於螢光素中的氨基酸35爲白氨酸以 外之氨基酸片段; h) 對應於螢光素中的氨基酸片段105爲丙氨 酸以外之氨基酸片段; i) 對應於汾似/7JTA75螢光素中的氨基酸片段234爲丁氨 二酸以外之氨基酸片段; j) 對應於螢光素中的氨基酸片段420爲絲氣 基酸以外之氨基酸片段; k) 對應於似;螢光素中的氨基酸片段310爲組織 氨基酸以外之氨基酸片段。 本發明的蛋白質包括 Lucj’oJa mj’ngreJica、Lucj’oJa crucj’ata 或酵素的螢光素序歹[],其中除了上述的位 置359有變異以外,至少還包括下列情況其中之一: a) 對應於螢光素的氨基酸356爲麩氨酸鹽以 外之氨基酸片段; b) 對應於螢光素中的位置215之氨基酸片段 爲一種除了丙氨酸與羥丁氨酸以外的忌水性的氨基酸;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如ϋ螢光素的片段216爲氨基乙酸(對於他 17 1239353 組成的序列)以外之氨基酸片段,或爲天冬素(對 Luciola cruciata或Luciola lateralis敏成的序讽來說从Js外之 氨基酸片段; 幻繫應、於 Luciola mingrelica、Luciola cruciata 或 Luciola 螢光素的片段234爲絲氨基酸以外之氨基酸片段; &)繫 Μ 於 LucioL· mingrelica、Luciola cruciata 或 Luciola 螢光素的片段297爲白氨酸以外之氨基酸片段; f) 截應、於 Luciola mingreiica 營光素的氨1 基駿 16、Luci’oh c/T/c/a々或价螢光素的氨基酸17爲苯基丙胺 酸以外之氨基酸片段; g) 繫Μ於 Luciola mingrelica 的片段 Υ!、或是:LucioL· cruciata 或的片段38爲離胺酸(lysine)以外之氨基酸 片段; h) 對應於 Zi/c/oAg 的氛基酸片段 106、 边的氨基酸片段107或/攸ϋ的氨基酸片段 108爲氨基乙酸以外之氨基酸片段;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 的氨基酸片段236爲氨基乙酸以外之氨基酸片段; Luciola mingrelica、Luciola cruciata 或 Luciola 的片段422爲羥丁氨酸以外之氨基酸片段;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 或 Luciola 的氛基酸片段312爲經丁氨酸(對/27//7^re/i^2 組成的序列來說)以外之氨基酸片段,或爲纈草胺酸(對、 Lucioia cruciata或LucwJa iaterahs組成的序列來說)以外之 18 1239353 氨基酸片段。 在任何一個有助於提昇熱穩定性的例子中,特定的取代 氨基酸可以由後續提到的標準方法來決定,在每一個情況 中,不同的取代都可能提昇熱穩定度,取代可以藉由在DNA 編碼本性的位置導向的變異,或是如熟習此技藝者知道的 適當的變異蛋白質來影響,本發明在此例子中係結合位置 的辨識,其中位置係與熱穩定相關聯。 通常無論如何都會想到以一個相對於天然的氨基酸具 有不同特性之氨基酸來作取代,因此親水性的氨基酸片段 可能在一些例子中較佳是以疏水性的氨基酸片段來取代, 而反之亦然,同樣的,酸性的氨基酸片段可以用鹼性的片 段來取代。 舉例來說,蛋白質可能包括一種具有螢光活性的蛋白 質、i 與 Photinus pyralis、Luciola mingrelica、Luciola cruciata 或酵素在酵素的序列上至少有60%的相似 性,至少包括下列情況之一: a) 對應於户加他以py/Ws螢光素中的片段214,或對應於 Luciola mingrelica ' Luciola cruciata M LucioJa lateralis 素的片段216之氨基酸片段有變異,且在 螢光素的例子中爲羥丁氨酸以外之氨基酸片段;或是 b) 對應於螢光素中的片段232,或對應於 Luciola mingrelica ' Luciola cruciata Luciola lateralis 素的片段234之氨基酸片段有變異,且在 螢光素的例子中爲異白氨酸以外之氨基酸片段;或是 19 1239353 C)對應於〃螢光素中的片段295,或對應於 Luciola mingrelica ' Luciola cruciata Luciola lateralis 素的片段297之氨基酸片段有變異,且比如在 ΑΚα/Α螢光素的例子中爲苯基丙胺酸以外之氨基酸片段; 而且相較於天然螢光素,這些螢光酵素具有增強的熱穩定 度。 所有各種螢光素的序列顯示這些螢光素高度保存在其 之間的相似性,這表示利用偵測大部分相似的區域檢查其 序列,可以很容易的決定這些酵素序列之中相對應的區 域,雖然爲了決定對應區域或在各種序列之間的特殊氨基 酸,可能會用到商用軟體(像是由University of Wisconsin Genetics Computer Group 提供的,’Bestfit” ;請見 Devereux 等 人在 1984 年的 Nucleic Acid Research 12: 387-395),此外或 者對應的酸可以參考L. Ye等人的Biochim. Biophys Acta 1339 (1997) 39-52 來決定。 在關於與螢光素的片段214相對應的氨 基酸片段方面,極性氨基酸羥丁氨酸適合被一個非極性的 氨基酸取代,像是丙氨酸、氨基乙酸、纈草胺酸、白氨酸、 異白氨酸、脯氨酸、苯基丙胺酸、蛋氨酸、色胺基酸,或 半胱胺酸。用來取代對應於仰0仏7以的片段214之 羥丁氨酸片段較佳是丙氨酸,而更好的是半胱氨酸。無論 如何,在這個位置上的不同的極性片段,像是天冬素,也 可以增加在此位置上具有羥丁氨酸的對應酵素的熱穩定 性;在天然的螢光酵素中,出現在這個位置上的其他氨基 20 1239353 锻包括氣基乙锻{Luciola mingrelica、Hotaria parvula、、天 冬素{Pyrophorus plagiophthalamus、GR、YG、YE或〇R、 Luciola Cruciata ' Luciola lateralis ' Lampyris noctiluca ' Pyrocoelia miyako Photuris pennsylvanica LY、KW、J19)VXSl 絲氨基酸,這些用非極性或不同非極性側鏈, 像是丙氨酸與半胱氨酸來取代都是有益的。 相較於在们价螢光素中,對應於片段232的 氨基酸片段的可能的改變,非極性氨基酸,異白氨酸,適 合被一個獨特的非極性氨基酸,像是丙氨酸、氨基乙酸、 纈草胺酸、白氨酸、脯氨酸、苯基丙胺酸、蛋氨酸、色胺 基酸,或半胱胺酸來取代,在天然序列中,出現在這個位 置的其他氨基酸包括絲氨基酸與天冬素。在/%<?///? iA? 營光素中繫應、於汽段2Ύ1,议及在Luciola mingrelica、Luciola 或/WWh螢光素中對應於片段234,較佳 用來取代的氨基酸片段爲丙氨酸。 改變在仰加“似螢光素中對應於片段295,以及 在仰6^/7似仍仰//?螢光素中對應於片段297的氨基酸片 段,也可以影響蛋白質的熱穩定性(在的螢光素 中則是對應於位置292),通常在此位置的氨基酸爲一非極 性的氨基酸,苯基丙胺酸或是白氨酸。用獨特的非極性氨 基酸來取代是適當的,舉例來說,在/¾如·77Μ 价螢光 素中,非極性的氨基酸,苯基丙胺酸,適合被一個非極性 的氨基酸取代,像是丙氨酸、白氨酸、氨基乙酸、纈草胺 酸、異白氨酸、哺氨酸、蛋氣酸、色胺基酸或半胱氨酸, 21 1239353 在户加如似螢光素中用來取代片段214的苯基丙胺 酸的較佳的氨基酸爲白氨酸。 在對應户加故似螢光素的氨基酸η,或對應 hc/仏螢光素的氨基酸16或17(在仰/7>〇你7·ζ螢光素中則 是13)的氨基酸片段也可能產生變異,此氨基酸片段(通常 爲苯基丙胺酸,但也可以是白氨酸、絲氨基酸、精氨酸或 是在一些例子中的酪氨酸)適合換成不同的氨基酸,特別是 一種獨特的非極性氨基酸,像是丙氨酸、纈草胺酸、白氨 酸、異白氨酸、脯氨酸、蛋氨酸或色胺基酸,較佳是丙氨 酸。 對應PAot/mJspjrWs螢光素的氨基酸35,或對應l—oh 螢光素的氨基酸37(在其他的族中則是 38)的氨基酸片段的變異也是有用的,此天然酵素在這個位 置上的各種氨基酸包括白氨酸但也可以是 離氨酸、組織氨基酸、氨基乙酸、丙氨酸、麩氨酸、以及 天門冬氨酸,在這個位置上的氨基酸片段適合用一種非極 性的氨基酸片段或一種不同飛機性的氨基酸,像是丙氨 酸、纈草胺酸、苯基丙胺酸、異白氨酸、脯氨酸、蛋氨酸 或色胺基酸來取代,在這個位置上最佳的氨基酸是丙氨 酸,這是於天然酵素不一樣的。 對應序列位置14的氨基酸,以及/或對 應於w 价螢光素氨基酸35的氨基酸片段的變異 較佳不僅是在酵素中有變異,也適合伴傭發生上述定義的 其他變異,特別是在對應於P加汾μ 螢光素位置 22 1239353 214、395或232的位置之變異。 街應PAotinus pyrai/s螢光素的片段Λ05,以及對應 Luciola mingrelica 營光素灼片段 、Luciola cruciata 營光 素的片段107或Ζπ/σΑ众化/你螢光素的片段108(在 中則是102)的氨基酸片段的改變也會影響蛋白 質的熱穩定性,通常在這個位置上的氨基酸爲非極性的氨 基酸,丙氨酸或氨基乙酸,或是絲氨基酸(在 中),適合用一種非極性的氨基酸來改變,舉例來說,在 中,非極性的氨基酸,丙氨酸,適合用一個 獨特的非極性氨基酸,像是苯基丙胺酸、白氨酸、氨基乙 酸、纈草胺酸、異白氨酸、脯氨酸、蛋氨酸或色胺基酸來 取代,其中較適合用來取代對應於螢光素 片段105的丙氨酸片段的氨基酸片段爲纈草胺酸。 對應户加如似螢光素的片段234,以及對應 Luciola mingrelica ' Luciola cruciata^ Luciola laterlis 素的片段236(在以中則是231)的氨基酸片段的改 變也會影響蛋白質的熱穩定性,通常在這個位置上的氨基 酸爲天門冬氨酸或氨基乙酸,而在一些情況中爲麸氨酸或 羥丁氨酸,其中用非極性或獨特的非極性氨基酸來改變是 適當的,舉例來說,在户加加以中,天門冬氨酸的 氨基酸片段適合用一個非極性氨基酸,像是丙氨酸、白氨 酸、氨基乙酸、纈草胺酸、異白氨酸、脯氨酸、蛋氨酸或 色胺基酸來取代,其中較適合用來取代對應於/¾⑽ 螢光素片段234的苯基丙胺酸片段的氨基酸片段爲 23 1239353 氨基乙酸;其中位於此位置的非極性氨基酸片段(以在 Zz/c/oh螢光素中爲例),像是氨基乙酸則可以用一個特殊的 非極性氨基酸來取代。 對應螢光素的片段420,以及對應 Luciola mingrelica ' Luciola cruciata^ Luciola laterlis 素的片段422(在綠色中則是417,而在 紅色中則是418)的氨基酸片段的改變也會影響蛋白質的熱 穩定性,通常在這個位置上的氨基酸爲不帶電荷的氨基 酸,絲氨基酸、羥丁氨酸、或氨基乙酸,適合用一種特殊 的不帶電荷氨基酸來改變,舉例來說,在乃加/λμ 中,絲氨基酸可以用天冬素、麩胺酸、羥丁氨酸或酪氨酸 來取代,且特別是羥丁氨酸。 對應PAodnus py/^//s螢光素的片段310,以及對應 Luciola mingrelica ' Luciola cruciata^L Luciola laterlis 素的片段312的氨基酸片段的改變也會影響蛋白質的熱穩 定性,在已知的螢光素蛋白質之中,這個位置的氨基酸分 规爲在 Photinus pyralis、Pyrocoelia miyako、Lampyris noctihica 以及 Photuris pennsyhnvanica 營光素的一鱼型態中 是組織氧基駿,在 Lucioia mingrelica、Hotaria parvula 以及 在此爲氨基酸307)螢光素中是羥丁氨酸,在 與 中爲纈草胺酸,而在一些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/叹/即价加/趟似螢光素中爲天冬素。因此,通 常在這位置上的氨基酸爲親水性的氨基酸,可以甩一些增 加酵素熱穩定性的特殊氨基酸片段來改變,特別用來在 24 1239353 螢光素中取代對應於片段310的組織氨基酸 片段較適當爲精氨酸。 其他的變異也可能存在酵素中,舉例來說,在一個較佳 實施例中,蛋白質也可以在用加“似螢光素的氨基 酸354(在螢光素中是356)的相對應的位置上由麩胺 酸的氨基酸改變,特別是變成氨基乙酸、脯氨酸或天門冬 氨酸以外的氨基酸,在此位置上適當的氨基酸爲色氨基 酸、纈草胺酸、白氨酸、異白氨酸或天冬素,但較適當的 爲離氨酸或精氨酸,此變異在WO 95/25798中有說明。由 此可以發現,在此位置上的忌水的片段會增進酵素的波長 位移,此外,在位置354上一個大的忌水的(V或I)、極性 (N)或是正電荷(K或R)的氨基酸的存在均會增進熱穩定性。 在一個替換的較佳實施例中,蛋白質也具有位置對應 Luciola螢光素(在中則是215)中氣基酸217 的氨基酸被改變成一個忌水的氨基酸,特別是異白氨酸、 白氨酸或纈草胺酸,如ΕΡ-Α-052448中的說明。 在本發明中的蛋白質包括天然的與重組的螢光酵素兩 種,其與天然序列至少有60%的相似性,像是如μ pyralis、Luciola mingrelica、Luciola cruciata 或 Luciola 酵素,就了解在天然酵素中至少有60%的氨基酸是 存在於本發明的蛋白質中,這類蛋白質可以有較高的相似 性,特別是至少有70%,較佳是至少80%,且更至少佔上 列的天然酵素的90%以上,這類的相似蛋白質包栝突變的 異變體(allelic variant),來自其他昆蟲種類的蛋白質’像是 25 1239353 重組產生的酵素。其中可以很容易的確認這些蛋白質是用 核酸(nucleic acid)來編碼的,這些核酸是在嚴格的雜交條件 下,以編成天然酵素的序列來混種而成,其中熟習此技藝 者很容易瞭解其中所指的條件,且可以在Sambrook等人於 1989 年所著的 Molecular Cloning (Cold Spring Harbor Laboratory Press)中所提的例子作爲例證。在一般的條件 中,低嚴格的條件可以被定義爲在環境溫度爲攝氏65度下 三倍的SSC,而高嚴苛的條件則爲攝氏65度下的0.1倍的 SSC,其中SSC爲緩衝溶液的名字,包括0.15M的氯化鈉, 0.015M的棒檬酸三鈉(trisodium citrate),三倍的SSC表示強 度爲爲SSC的三倍。 特別的是,本發明序列的一種特定序列的相似性可以用 多重的對準方式來評估,此方式係由Lipman與Pearson提 出的(Lipman,DJ. & Pearson,W.R· (1985) Rapid and Sensitive Protein Similarity,Science,vol 227, ppl435-1441)。,, 樂觀的”百分比範圍應該可以用下列用於Lipman-Pearson算 法的參數來計算:ktup =1,gap penalty =4 而 gap penatly length =12,被評估用於相似性的序列應該可以用以作爲” 測試序列”,此表示其爲對照的基本序列,像是 AKra/Zi?的序列或是由Ye寺人紀錄的其他序列中的任何—^ 個,都可以被帶入上述的演算法中。 本發明的蛋白質的特定例子爲具有一或多處變異的天 然螢光素序列,如上所述。 本發明進一步提供編譯成上述的螢光素的核酸,其中核 26 1239353 酸係爲習知的天然序列的基本,基於基因密碼的知識,適 當的變異會造成氨基酸序列預期的改變很容易表現出來。 在本發明的一個較佳實施例中,核酸是一種合成的基 因,合成的基因被設計用來移除僅在重要的顯性基因 (expressed genes)中發現的密碼單位,這些顯性基因係取自 普通基因特質宿主像是五cW/,而且同時可以避免僅在甲 蟲的基因編碼中發現的密碼單位的傳入,此方式可以確保 因的基因具有一個密碼單位的利用,這均適用於五與 昆蟲的表現系統。 舉例來說,無論用於氨基酸的密碼單位,arg、leu、ile、 gly與pro是否有可能改成CGT或CGC(arg)、CTG,CTT或 CTC(leu)、ATC 或 ATT(ile)、GGT 或 GGC(gly)、以及 CCG,CCA 或CCT(pro),都會因此刪掉稀少的密碼單元。在下列敘述 與第14圖中說明的合成基因的例子中,結果有總共139靜 止的變異產生62新的非稀有(non-rare)的密碼單元(佔全部 的11%),在第14圖中所示的前8個核甘酸形成部分的核醣 體連接點,因此沒有指定密碼,編碼序列由用向上箭號表 示的蛋氨酸片段開始,此編碼序列與本發明的一較佳實施 例的相似序列,比如具有至少90%相似性的序列,或較佳 爲至少具有95%相似性的序列相近。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.
另外一個有用的特徵爲新的獨特的限定位置的結合,這 可以使用在產生一個合成集合時,這些位置較簡單且較有 效率的在基因上產生突變基因,特別是組合的匣式突變基 因(cassette mutagenesis),特別可以適合在酵素中的次領域B 27 1239353 的cDNA編碼之間創造獨特的限定位置,在基因的端點3, 處額外的產生一個獨特的限定位置,有助於允許簡單的融 合,以及/或移除過氧晦體目標序列。 在此後介紹的例子裡面,有九個新的獨特限定位置被言受 計出來,大部分是在基因的中間第三的位置,且一個獨特 的後三位置在基因的端點3’處形成,以允許簡單的C-端窯占 融合(見第12圖)。 最後,使用合成的基因可以用於產生變異,以增加基因 產物的熱穩定性,或在其他方面修正其產物的特性,在此 後敘述提到的係中,三個非靜止的變異被用來產生具有熱 穩定性的氨基酸轉換T214C、E354K與D357F變成多胜 (polypeptide) ° 本發明的核酸適合與顯性的載體(vector)結合,像是在 控制單元,像是促進子(promoter)、增進子(enhancer)、終結 子(terminator)等控制下的細胞質體,這些載體可以接著被用 來傳遞一個宿主細胞,比如一^個原核(prokaryotic)或真核細 胞像是植物或動物細胞,特別是在一個原核細胞像是丑 中,所以細胞可以表現出預期的螢光酵素的特性’像這樣 的傳遞細胞培養造成螢光酵素的生產,其使用條件是熟習 此技藝者皆知的,這些螢光酵素接著可由培養媒介中分離 出來,當細胞爲植物或動物細胞時,可以用這些細胞來繁 殖植物或動物,接著可以由這些植物萃取出蛋白質’或在 轉換基因的動物中,蛋白質可以由奶中重新取得’載體、 轉換型態的細胞、轉換基因的植物與動物,以及利用培養 28 1239353 這些細胞產生酵素的方法均包括在本發明的進一步目的 中。 在下面接著說明利用隨機變異產生处的· 變異螢光素,其中發現D357Y的單一點變異 在放射光的波長中產生一個很大的顏色位移,而且相較於 天然的螢光素,也具有很大的熱穩定性,進一步的硏究顯 示在此位置的某個範圍的取代可以提昇到一個好的熱穩定 度,且/或有大的顏色位移。 汾變種酵素的特殊例子,落入本發明的範 圍中的如下所述: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:
D357YD357Y
D357FD357F
D357WD357W
D357KD357K
D357ND357N
D357ID357I
E354I/D357YE354I / D357Y
E354V/D357YE354V / D357Y
E354C/D357YE354C / D357Y
E354R/D357YE354R / D357Y
E354S/D357YE354S / D357Y
E354N/D357YE354N / D357Y
E354K/D357ME354K / D357M
E354R/D357L 29 1239353E354R / D357L 29 1239353
E354W/D357WE354W / D357W
E354H/D357WE354H / D357W
E354R/D357FE354R / D357F
E354K/D357FE354K / D357F
E354S/D357FE354S / D357F
E354M/D357FE354M / D357F
E354A/D357RE354A / D357R
E354A/D357FE354A / D357F
E354T/D357YE354T / D357Y
E354A/D357NE354A / D357N
I351M/E354R/D357VI351M / E354R / D357V
E354S/D357VE354S / D357V
E354R/D357WE354R / D357W
E354R/D357ME354R / D357M
E354R/D357SE354R / D357S
E354N/D357S 或是當由其他昆蟲的螢光素中取得時,與這些之任一的相 等物。 利用定位之變異基因、(PCR)或組合的匣狀變異基因, 將用來產生上述變化的變異導入在細胞質體pET23上的螢 光素基因中,將寡核甘酸(oligonucleotide)加入PCR反應中, 以產生關聯性的變異,如下所述。 如前面提到的在354與215位置上的點狀變異的影響是 附加的,本發明提供了組合三或更多種變異的可能性,以 1239353 在具有大的顏色位移的變種酵素中提供高的熱穩定性。 本發明之螢光素蛋白質在生物發光分析時很有用,其乃 利用螢光素(luciferase/luciferin)反應作爲一個訊號物件,有 很多這類的分析可見於文獻中,蛋白質因此可以被包括在 準備用來作爲這些分析的試驗組(kit)中,選擇性的與螢光素 (luciferin)以及其他用於進行特定分析所需要的試劑合倂使 爲讓本發明之上述目的、特徵、和優點能更明顯易懂, 下文特舉較佳實施例,並配合所附圖式,作詳細說明如下: 圖式之簡單說明: 第1圖爲一個對數圖,顯示本發明中在45°C下,幾種 培養的變種酵素其剩餘活性百分比與時間的關係; 第2圖爲光譜圖,由具有螢光酵素的培養的細胞 取得,此細胞係在具有D-螢光素的檸檬酸鹽緩衝溶液中, 其中使用的酵素爲(a)重組的天然凡(9价以蚤光素、 (b)—種D357K的變種、(c)一種D357N的變種、(d)—種 D357W的變種、(f)一種D357F的變種、(g)—種D357Y的變 種、以及(h)—種E354I+D357Y的雙重變種; 第3圖爲一個顯示三種變種酵素,E354I、D357Y以及 E354I/D357Y雙重變種(DM),的剩餘活性百分比與時間的關 係的圖表; 第4圖爲一個放射光譜,包括⑻重組天然酵素,以及(b) 雙重變異(DM) E354I/D357Y ; 第5圖爲一個光子放射衰減率的圖表,包括一個重組的 31 1239353 天然酵素(♦)r-wt以及一個D357K變種酵素(j); 第6圖顯示一個分子模型圖,說明一種在螢光酵素中可 能的CoA連接開口; 第7圖顯示在活的有機體中,由Coli細胞放射的生物 發光光譜,此細胞表現出變種的A Aj⑽价營光素D357Y(a) 在LB上成長;(b)在極小的媒介與醋酸鈉上成長;(c)在極 小的媒介以及葡蔔糖上成長; 第8圖顯示在活的有機體中,由Coli細胞放射的生物 發光光譜,此細胞表現出變種的户.螢光素 E354K/D357M(a)在LB上成長;(b)在極小的媒介與醋酸鈉 上成長;(c)在極小的媒介以及葡萄糖上成長; 第9圖顯示一個利用户.螢光素D357Y的突變, CoA在放射光的光譜貢獻上的影響之圖表·, 第10圖顯示一個利用A 价螢光素D357Y的突變, CoA在放射光的光譜貢獻上的影響標準化的資料圖表; 第11圖顯示一個利用Pura价螢光素E354I/D357Y的 突變(第11a圖),CoA在放射光的光譜貢獻上的影響之圖 表,以及標準化資料(第lib圖); 第12圖說明在一個合成的螢光素基因架構中利用到的 限定位置變更; 第13圖說明在一個螢光素基因的合成中使用的架構; 第14圖顯示合成螢光素基因的cDNA之序列(SEQ ID NO 1)(包括構成部分核醣體連接單元但並未編碼的核甘酸 1-8),以及由向上的箭頭標示出的蛋氨酸片段開始編碼的氨 32 1239353 基酸序列(SEQ ID NO 2);以及 第15圖說明在50°C下由合成基因編碼的變異之熱穩定 性。 第一實施例 變種螢光素的證明與特件鑑定 準備由錯誤傾向的(error-prone)PCR[M· Fromant et al·, Anal· Biochem· (1995) 224,347-353]建立的關於螢火蟲 螢光素的兩個基因段,其中一個基因段包 括完整長度基因的錯誤傾向的PCR產品,在T7顯示系 統 pET23a 中複製(Novagen Inc·,Madison,WI,U.S.A),第二 個基因段包括/π基因的一個短片段的錯誤傾向的PCR產 品,包括氨基酸199-352,在載體pBSK(+)中複製出來的 (Stratagene,La Jolla, CA,U.S.A.) 〇 pET23a基因段被表現在五系統BL2UDE3)中, {E.CoIi B Y dcm ompT hsdS(rB· ιώβ·) gal又(Χ>Έ3))。 pBSK(+)基因段被表現在HB101細胞中,(仰 aral4 galK2 lacYl A(gpt-proA)62 rpsL20 (Strr) xyl-5 mtl-1 red/J」細/rC-mre 价W 。pET23a 與 pBSK(+)兩者帶 有β-lactamase的基因,且使帶有細胞質體的万.細胞有 安比西林的抵抗力。 一個万.6^//系統與利用electroporation製備的基因段一 起被轉換,使用一個BIORAD五π/y脈衝器,於37°C的LB 洋菜上成長整夜,其含有的安比西林濃度爲50μβ/πι卜細胞 被送到尼龍的薄膜上(Osmonics,Minnetonka,Minnesota, 33 1239353 U.S.A·),而且噴灑上螢光(luciferin)溶液(500μΜ的D-螢光 素,鉀鹽,在lOOmM的檸檬酸納緩衝溶液中,pH5.0)。此 微生物的繁殖(colonies)可用 AlphaImagerTM 1200 Documentation and Analysis System (Flowgen,Lichfield, Staffordshire,UK)來觀察,利用微生物的繁殖透過一段時間 會集結放射出生物發光,而產生光放射的影像,光的亮度 可以作爲螢光素熱穩定性的一個指標。 接著檢測此微生物的熱穩定性,在放射光的亮度基準上 篩選此微生物,且進一步利用其特性加以區隔,在一些檢 測中,五的微生物在檢測之前會在42°C下先被培育兩 個小時,所以熱穩定的變異可以被挑出來。由初步檢測區 隔出來的微生物會被暫時的塗貼在尼龍薄膜上,且在含有 安亞西林的LB媒介中成長整夜,這些塗貼物會被噴灑上螢 光溶液,並在Alphalmager™中觀察;進階的檢測有助於在 試管分析中清楚的確認微生物的螢光活性,可能具有熱穩 定酵素的五的微生物會在試管中分析其熱穩定性與螢 光活性。 在試管中進行螢光活性分析的步驟係在室溫下進行,使 用 Promega 螢光素分析系統(promega Corporation,madison, WI,U.S.A·)。 利用將1〇μ1的天然細胞萃取液加入到1〇〇μ1的Promega 螢光素分析溶液(cocktail)(l對2稀釋)中開始螢光反應,利 用Biotract M3亮度測量儀來量測結果產生的生物發光狀 態。 34 1239353 天然細胞萃取液的製備在Promega技術期刊的第101期 中有提到,整夜培養的整數的五cW/被溶解在細胞培養溶 解試劑中,其中細胞培養溶解試劑包括25mM,pH7.8的三 憐酸鹽、2mM 的(dithiothreitol,DTT)、2mM 的 1,2-雙氨基環 己 烷 -N,N,N’,N’- 四 乙 酸 (l,2-diaminocyclohexame-N,N,N’,N’-tetraacetic acid)、10%的 丙三醇、1%的氚核X-100、1.25mg/ml的hen溶菌酵素,在 室溫下反應10分鐘,接著,在進行分析之前將天然的lysate 儲存在冰上。 在與時間相關的去活性硏究中檢測酵素的性質,裝有 50μ1的完整天然細胞萃取液的Eppendorf試管會被放置在具 有一個給定溫度的水中培養,在設定的時間點將試管移 出,並在分析前先以冰冷卻,將剩餘的螢光活性用原來螢 光活性的百分比來表示。 剩餘活性百分比與時間的關係會以對數表示作成圖 表,並用來計算T1/2的値,T〃2表示在指定的溫度下培養, 酵素損失50%的原來活性時所需花費的時間,由剩餘活性 百分比對時間的對數圖,來決定在37°C下天然的萃取液的 Tw2値(活性減低到原來活性的50%時的時間)。 由上述具有大部分熱穩定螢光素的五微生物中的 細胞質體DNA會被排序,以決定與酵素的熱穩定性有關的 變異。 利用 QIAGEN QIAprep Spin Miniprep Kit,(QIAGEN Ltd, Crawley,W. Sussex, UK),接著根據微離心分離機的操作方 35 1239353 法(QIAprep Miniprep Handbook 04/98),就可以製備細胞質體 DNA。 透過 Babraham Technix,Cambridge, UK,根據 dideoxy chain termination 的方法,使用一種 ABI PRISMtm 377 DNA Sequencer Ready Reaction Kit (Perkin Elmer Applied Biosystems),可以決定所有的DNA序列。 此做法的結果可以確定一個新的變異D357Y。 螢光素的結晶結構[E. Conti et al·,Structure,4 (1996) 287〜298]顯示位置357係位於蛋白質的表面上,且接近位置 354,可以影響熱穩定性與光譜性質,這表示此區域對酵素 的熱穩定性很重要。 D357Y爲一個獨特的熱穩定變異,變成最穩定的螢光 素,其中有一個氨基酸改變。 實施例2 單元指向的發生變異以創浩其他的357變異 爲了評估在357位置上不同的變異,定位(site-directed) 的變異(mutagenesis)會被進行,進行的方式係使用Stratagene QuickChange™ Site-Directed Mutagenesis Kit, (Stratagene, La Jolla,CA,U.S.A.),其中細胞質體 pPW601a J54,(PJW,MoD Report, 3/96)會被用在所有的單元指向的發生變異中。所有 變異反應的產物會被送到五從//系統XLl-Blue中, [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 ΖΛΜ15 Τη 10 (Tef) Amy Camr]] j 利用 Sigme-Genosys Ltd·,Cambridge,UK 合成,並利用一個智慧 的植入系統[A.R Arkin et al·,Biotechnology,(1992)10, 297-300, W,· Huang et al·,Anal. Biochem· 218, 454-457]來設 計寡核甘酸的原始質(oligonucleotide primers),以產生較使 用每一氨基酸取代的個別的原始質更有可能產生變異的族 在此方法中,會作出氨基酸取代螢光素的變異基因段 組。 接下來會使用到寡核甘酸(以及其互補的部分):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’) 氨基酸取代 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 變異的基因段組可以對熱穩定性預先甄選出來,被測試 的微生物的數量可以利用公式計算[S. Climie et al·,L Biol· Chem. 265 (1990) 18776-18779] N = [ln(l-P)]/[ln((n-l)/n)] 其中N爲被檢測的微生物數量,n爲在目標位置可能的密 37 1239353 碼單位的數量,而p爲在混合物中的每一個密碼單位備取 作樣本至少被檢測一次的機率,計算係根據Ρ=0·95來進 行,由單位指向發生變異得到的變異會被分析,在與時間 相關的熱去活性硏究中分析螢光素的活性與特性。 用此方法確定預期的突變會在400毫升含有安亞西林 的LB媒介中成長到Α260«0.5,接著加入異丙基β-硫基半乳 醣甘(isopropyl β-thiogalactoside,IPTG)達到最終濃度 ImM, 以產生螢光素的表現,此細胞接著在30°C下震動培養達三 個小時,然後再以離心分離基收集,結果的細胞九會懸浮 在10毫升ImMDDT的B-PER™蛋白質萃取試劑中(Pierce Chemical Company,Rochford,U.S.A·),以生成一個天然的萃 取物,接著進行用於 Maxi-Scale Bacterial Protein Extraction 的B-PER™方法,將500μ1的再製備Sigma蛋白晦抑制劑溶 液(Product No. P8465,Simga,Saint Louis,Missouri,U.S.A.) 加入到B-PER™溶液中,以抑制內部生成的蛋白晦,此細胞 溶解素接著會在30000克下被離心30分鐘。 利用硫酸錢來分餾浮在表面的天然的萃取物,沉殿在 30%與55%之間飽和的分餾物含有螢光活性,此材料會懸 浮在0.5毫升ρΗ8·0,ImM DTT的Tris氯化氫中,並用於熱 去活性與光譜的硏究上。 進行D357L,T,V,W,R,I,S,Κ,N與F的取代,這些變 異會在試管中進行天然的萃取物的熱去活性的硏究中被依 其性質分辨。 特別純化的萃取物經過1比11的稀釋,加入到一個熱 38 1239353 去活性的緩衝溶液中:50毫升的磷酸鉀緩衝溶液,ρΗ7·8, 含有10%飽和的硫酸銨、ImM的dithiothreotol以及0.2%的 BSA 〇 在40°C或45°C下設定時間週期來培養11〇μ1的完整蛋 白質溶液,然後在分析之前用冰加以冷卻,接著如實施例1 所述,使用Promega螢光素分析試劑(1對2稀釋)來量測其 螢光活性。 結果顯示在表2與表3以及第1圖中,Tm的値會由在 40°C (表2)與45°C (表3)下天然的萃取液來決定。 表3 變異 Tl/2 D357W 2.5 D357F 6.5 D357Y 10.4 RWT <1.0 表2 變異 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 相較於重組的天然型態,所有的取代都顯示可以增進熱 穩定性。 實施例三 39 1239353 放射光波長的改變 在位置357的氨基酸取代也可以觀察到,對在試管中的 酵素放射出的光譜有影響,一個完整(250μ1)的的微 生物,如實施例二所述係在37°C下整夜成長,然後在一個 微離心分離機中旋轉,並將懸浮物去掉。表現不同變種螢 光素的細胞會在一個含有150μ1的D-螢光素的檸檬酸鹽緩 衝溶液(ΡΗ5.0)中培養,而且透過測量其放射光譜,來分析 由活的有機體反應中放射出的光,測量係使用 SPECTRAmax® Microplate Spectrofluorometer(Molecular Devices Corp· California,U.S.A·)來進行。突變 D357Y,F 與 I(請見圖2(aHg))可以由光譜峰就像波長有大的改變來觀察 到,這些結果摘錄於下列的表四中。 此外,在活的有機體中的變異之光度在暗室中用肉眼就 可以判斷,D357的變種在色度光譜上顯現出一個多樣化的 顏色,特別的是D357Y,F與I在放射光上表現出明顯的長 波長位移。 在一些情況中,比如D357F,比入放射光顏色出現適當 的改變,不僅是波長λΜΑχ有位移,也會因爲不同的可見光 波長而對光譜產生不同的貢獻。 重組天然的(r-wt)酵素會被用來與在活的有機體中一些 357發生突變的放射光波長λ MAX作對照,D357Y,F與I在 波長的最大値上呈現一個理想的位移。 1239353 表四 變異 λ MAx(nm) rWT的偏差(nm) rWT 558 D357K 556 -2 D357N 558 0 D357W 558 0 D357I 606 +48 D357F 611 +53 D357Y 613 +55 實施例四 有或沒有CoAJ#的酵素特性 如實施例一中所述,利用硫酸銨沈澱來部分純化 D357Y,將部分純化的D357Y酵素(5μ1)與150μ1的Promega 螢光素分析試劑混合,另外一部份與一等量的分析緩衝溶 液混合,緩衝溶液中沒有CoA(25mM的Tristricine、pH7.8、 5.0mM 的硫酸鎂、O.lmM 的 EDTA、2mM 的 DTT、470μΜ 的 D-螢光素與530μΜ的ΑΤΡ),測量兩個反應的放射光譜,並 繪示於第9圖與第10圖。 在沒有以及有CoA情況下,光譜顯示生物發光放射一 個標示的不同處,一個明顯的位移λ MAX。在螢光反應的動 力上,CoA的效果也可以由在RLU基準中的不同來觀察到 (relative light units,RLU表示相對的亮度單位)。 放射的不同使在分析偵測CoA的存在中使用酵素的可 1239353 能性增加。 實施例五 雙重變異的製備與其特件 如實施例二中所述使用定位的變異,爲了硏究在熱穩定 性與放射光顏色上的任何累積效果,而設計了 E354I+D357Y 的雙重變異物。 將部分純化的雙重變異物,E354I+D357Y,以1對11 稀釋爲一個熱去活性的緩衝溶液:50mM的磷酸鉀緩衝溶 液,PH7.8,含有10%的飽和硫酸銨、ImM的dithiothreitol 以及0.2%的BSA 〇 在45°C下將ΙΙΟμΙ的完整蛋白質溶液培養一段時間,然 後在分析之前以冰冷卻之,接著使用Premega螢光分析試 劑(1對2稀釋),像前面一樣量測其螢光活性。 相較於個別的單一變異物E354I與D357Y,雙重變異物 在熱穩定性上呈現了一個明顯的增進效果(如第3圖所 示),在一個給定的Tm値爲7.7分鐘,下當在45°C下去活 性,部分純化雙重變異物的熱去活性硏究證實了變異物熱 穩定性的增加。 在此須注意,相較於個別變異物E354I與D357Y,雙重 變異物呈現一個非常深紅色的螢光,在螢光炎熱上有加成 的效果。 重組天然種類與雙重變異物E354I+D357Y天然的萃取 物放射光譜都可以使用在實施例3中提到的分析緩衝溶液 42 1239353 來進行量測。 在活的有機體中,量測的放射光譜給了一個611nm的 λ max,無論如何,波長爲紅光區域的光譜在發光上有一個 更大的貢獻’導致肉眼看來呈現很深的紅色;在天然的萃 取物的放射光譜在光譜的形狀上呈現一個明顯的改變,而 且相對於rWT,其波長位移爲44nm(請見第4圖)。 在活的有機體中,雙重變異物的放射光譜顯示放射光的 波長峰(613nm)的帶寬變的尖銳,且在範圍540-560nm的放 射光波長的貢獻降低。 這些變異顯著的影響表示在酵素的此區域,對生物發光 性的光線之重要性。 實施例六 改進的光子磁力 在活的有機體中,相對於這個位置的其他變異物,表現 D357K變異物的及細胞的生物發光是很亮的,此酵素 的燃燒動力(flash kinetics)可以用一個照度計來分析,此照 度計可以量測光子對時間的放射速率。含有重組天然酵素 或變異物D357的整個五Cb/人細胞任意萃取物會被佳到一個 螢光分析溶液中,此溶液中不含有任何會促進發光動力的 試劑,像是輔膊A,量測光子放射對時間(15秒)的衰減率, 可以看到變異物D357K有很明顯的減緩效果(如第4圖所 示)。在其他方面,變異酵素具有反應動力,至少在第一個 15秒的反應中,會較重組天然酵素被抑制到一個較小的程 43 1239353Oligonucleotide 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
實施例七 存E354與D357的組合匣式變異反應 步驟一 匣式變異反應的細胞質體pPW601aJ54的設計 在細胞質體pPW601a/J54中,使用兩對合成的寡核甘 酸,將兩個新的獨特的限定單元導入到he基因中(如下), 六個靜止的變異全部導入基因中的一個Spel與一個ΚρηΙ 限定單元,63基準對部分。細胞質體包含這些稱爲 pPW601aJ54SpeI/KpnI的新單元,這些限定單元的存在與接 近會使其可能使用組合匣式辨識,以探究在螢光重螢光素 的初級序列中,氨基酸位置357與357的隨機變異的影響。 Spel (a) 5?-gggctcactgagactacTAGTgctattatgattacacccg-3' ntlQ21- nt!060 (SEQ ID NO 8)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)Spel (b) 5'-cgggtgtaacagaatagcACTAgtagtctcagtgagccc-3 (SEQ ID NO 9)
Kpnl (a) 5'-ggcgcggtcggtaaagtGgtAccattttttgaagcg-3 ntl078- ntlll3 (SEQ ID NO 10)Kpnl (a) 5'-ggcgcggtcggtaaagtGgtAccattttttgaagcg-3 ntl078- ntlll3 (SEQ ID NO 10)
Kpnl (b) 5'-cgcttcaaaaaatggTacCactttaccgaccgcgcc-3? (SEQ ID NO 11) 44 1239353 在核酸膽的辨識單元,以及那些在上述情況中點的突變 發生的位置中,很明顯需要核甘酸來產生此單元。 步驟二 匣(cassette)的設計與某丙構 當回火(anneal)產生一個雙重獨立的匣時,此匣可以被 直接縫合到在新的限定單元吸收的細胞質體 pPW601aJ54SpeO/KpnI中,一對合成的寡核甘酸會被合成出 來,此匣被設計用來在初級序列的位置354與357中,導 入20個天然產生的氨基酸所有可能的組合。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)Looplib2A 5,-ctagtgctattctgattacacccNNG / CggggatNNG / Caaaccgggcgcggtcggt aaagtggta-3 ’(SEQ ID NO 12)
Looplib2B 5’- cactttaccgaccgcgcccggtttG/CNNatccccG/CNNgggtgtaatcagaatag ca-3’ (SEQ ID NO 13) 每一個迴路基因段的寡核甘酸取2μ2,與一個含有 50mM的Tris氯化氫、pH7.4、25mM的氯化鈉的緩衝溶液混 合,且在100°C下加熱3分鐘,此溶液接著在一個熱區塊小 於50°C下緩慢的冷卻,以回復到完整的序列,回火的寡核 甘酸接著被縫合到細胞質體pPW601aJ54SpeI/KpnI中,此細 胞質體已經被Spel與ΚρηΙ吸收了。用electroporation接著 將整個縫合反應用來轉換五HB101細胞’在在 45 1239353 electroporation以後,轉換的細胞會被塗在含有50pg/ml安 比西林的LB洋菜板上,並且在37°C成長整夜。接下來的 日子869繁殖的微生物會由板子上被隨機採收下來,且用 來在接種到1毫升含有安比西林的LB的一個96四方板 (Beckman)中,板子被覆蓋而細胞在37°C搖動下成長整夜。 步驟三 在活的有機體中檢測隨機取樣的微生物 在接下來的清晨中,將50μ1的整夜培養固定狀態的整 個物體傳送到兩個乾淨的塑膠圓板(Dynex)中,其底井爲96 微脂酸固定度(microtitre),其中一個板子會被覆蓋,且在一 個加熱的區塊上培養8分鐘(區塊的表面溫度爲45°C),而 另外一個則維持在37°C。接著檢測與紀錄在活的有機體 中,取自兩個板子的細胞中的螢光活性,檢測係在室溫下 進行,藉著將PH5.0,含有0.5mM的D-螢光素的50μ1具有 100mM的檸檬酸鈉緩衝液加入到井中,並接著將板子傳送 到一個影像攝影捕捉系統(Alpha Imager)中,透過加熱與控 制微生物群放射的光會被積集1或2分鐘,然後將影像紀 錄在熱感膜上。 在第二輪的檢測中選出呈現最佳的生物發光性的七十 九個微生物群,最佳發光性係由紀錄在膜上的影像亮度來 決定,這次這些微生物群在分析之前會被放在加熱區塊上 培養16分鐘。在由活的有機體中熱穩定測試選定的55個 無性繁殖個體中,25個會被選來進行活的有機體之光譜分 46 1239353 析’這些無性繁殖個體會在37°C下於LB中成長整夜,且 在接下來的清晨中取200μ1進行離心分離,然後將五 細胞九放置在一個白色塑膠微脂酸固定度板子中,使用一 種 Molecular Devices Spectramax 96 well plate Huorimeter 來 分析在活的有機體中由每個變異螢光素的發光放射光譜, 其結果被摘錄於上述的表一^中。 步驟四 確認變異 使用基因特定排序原始値來排序微生物,且利用在活的 有機體中檢測選定25個微生物,由其中製備細胞質體的 DNA,在初期排序中位置354與357改變的氨基酸中的變異 結果可以被確認,其中有一種變異也包含在位置1351的一 個氨基酸取代之一種額外的變異結果,請見表五。 1239353 表五 變異酵素 變異處 波長的峰(nm) 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/D357Y 612 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 其中rWT表示重組的天然型態。 由活的有機體中熱穩定度的分析選出一系列的變異螢 光素’大多數的螢光素在活的有機體放射光光譜上中也有 很大的變化’很多都在往光的長波長(>580nm)上有大的貢 獻’一些光譜也顯示在610_614nm的一個單峰的帶寬有明 顯的變窄。 E354與D357的一個忌水性的取代,以及使用一個芳香 族氨基酸’分別像是E354V、D357Y,會在活的有機體之光 譜上有最大的改變,約在612nm處出現一個具有窄的帶寬 鲁 的單一吸收峰。 實施例八 - 查試管中檢J[[熱穩定件 利用溶解來製作選定微生物的細胞萃取物,且在一個熱 的去活性實驗中決定每一個萃取物的營光素之熱穩定性, 將50μ1的每一種萃取物放置在一個eppendorf試管中,並在 45°C的水域下培養4, 9與16分鐘,在適當的時間點將整個 φ 移開’且測量其剩餘活性,表六表示所有的變異酵素與重 組的天然種類相比,剩餘活性百分比對時間的關係。 49 1239353 表六 酵素編號 在45°C下培養以後剩餘活性百分比 (見表五) 0分鐘 4分鐘 9分鐘 16分鐘 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 其中nd表示沒有進行(not done)。 50 1239353 結果顯示大部分熱穩定的螢光素就是那些在位置357(Υ, F或W)上具有芳香族氨基酸,且在位置354上有一個大的 忌水的氨基酸(V或I)、極性的氨基酸(N)或正電荷(K或R)。 實施例九· 成長擐谙對活的有機體之放射光光譜的影響 由表現變異螢光素D357Y或E354K+D357M(表五中的7) 的丑a//BL21(DE3)細胞放射光光譜上,不同的碳源的影響 將會被分析檢測。 在LB媒介的中央木材相(log phase)上培養50毫升的細 胞族群,然後以離心分離機收取,細胞九會再懸浮於1毫 升的無菌的蒸餾水中,且取出1〇〇μ1的懸浮物用來接種在位 於25毫升的Sterilin試管中的5毫升的新鮮LB,Μ9最小媒 介+2mM的檸檬酸鈉,或M9最小媒介+2mM的葡萄糖上, 這些族群會在37°C且搖動的環境下繼續成長,經過90分鐘 (D357Y)或是120分鐘(酵素7)以後,將200μ1的細胞培養物 離心取出,並再懸浮於150μ1的緩衝液中,此緩衝溶液爲 ρΗ5·0,含有0.5mM的D-螢光素的100mM的檸檬酸鈉緩衝 液。再懸浮的細胞接著被放置在一個微脂酸固定板中,且 利用 Molecular Devices Spectramax 96 well plate fluorimeter 來分析在活的有機體中,每一種變異螢光素放射的生物發 光放射光譜,其結果如第7圖與第8圖所示。 結果顯示由一個豐富媒介(LB)(第7a、8a圖)到一個有醋 酸鹽(第7b、8b圖)或葡萄糖(第7c、8c圖)作爲碳源的特定 51 1239353 最小媒介,其放射光往長波長位移的轉變,且較短波長的 影響會減少。 範例+ 重組的天然種類與變異螢光素的純化與其螢光特性 爲了分析輔因子輔膊A在生物發光反應的光譜上的影 響,重組的天然酵素與變異的螢光奉D357Y 以及E354I+D357Y會被純化成同種,結合一個143氨基酸 碳水化合物鍵結模型(CBM),可以自厭氧菌/Vrwyca 可以純化出這三種螢光素,這個CBM在選擇性連接酸的膨 脹的細胞膜質時有提到,且可以溶解的碳水化合物,乳醣 甘(galactomannan)與葡萄糖甘(glucomannan),會構成簡單的 單步驟同種純化的基準。 融合到CBM的螢光素可以與在天然的細胞萃取液中的 細胞膜質相鍵結,以溶劑清洗,接著利用可溶解的多醣類 來洗淨,用這個方法傳話的融合蛋白質會被用在分析中, 以測量在含有不同量的輔晦A的反應中放射光的波長,酵 素(5μ1)會被加入ΙΟΟμΙ的分析試劑中,此分析試劑爲25mM 的 Tris-tricine、pH7.8、5.0mM 的硫酸鎂、O.lmM 的 EDTA、 530μΜ的ATP以及470μΜ的D-螢光素,其中含有不同量的 輔晦Α。第9圖至第11圖顯示,增加輔膊Α的濃度對由純 化的螢光素D357與E354I+D357Y放射的光之放射光譜的影 響。 在活的有機體中分析來自五細胞的生物發光的放 52 1239353 射光光譜,其中細胞中含有融合到黴菌的CBM的C-終點的 螢火蟲螢光素,此分析並未顯示其與天然螢光素相近的細 胞有任何明顯的不同,在試管中分析由一種商業來源的純 化重組螢光素(Promega)的生物發光的放射光光譜,與融合 蛋白質放射之光譜相似。 因此可以判斷顯著的不同係與CoA的濃度有關,當輔 膊A的濃度增加時,光譜貢獻會改變,且在最高的CoA濃 度時,光譜會被590-630nm之中在610nm的一個顯著吸收 峰所影響,雙重變異對此光譜位移的影響最爲明顯,其中 在波長爲610nm處有一個明顯且窄帶寬的單一吸收峰(見第 11 圖)。 實施例+ — 製作在214C/354K/357F處有變異的合成的7¾加//7似价 螢光素 利用上面敘述中提到的合成技術,由寡核甘酸對設計與 組合一個合成的luc基因,設計此基因序列以形成一個具有 氨基酸214C、254K與357F的螢光素。 由Sigma-Genosys Ltd合成29對的重疊合成寡核甘酸, 由PAGE純化,且以約爲550bp縫合在三個接合處(IDRIS 1, 2 & 3,見第13圖),接著將每一個接合處分別與載體pBSK(+) 接在一起,且將最後的結構用來改變丑XLl-Blue細胞。 _含有接合的插入物之無性繁殖個體來製備細胞質體 DNA,且加以排序以確認〇RF的螢火蟲。在接合處(n-1)個 寡核甘酸的存在(寡合成的副產物)會使架構變的複雜,確認 53 1239353 唯一正確的接合IDRIS 2與PCR的DNA排序會被用來確認 IDRIS3的一種接合,其中IDRIS3在架構的5’端有一個基對 (base pair)被刪除,完整的ORF的接合則由連接一個含有 IDRIS1與IDRIS2及3的細胞質體混合物來完成。 接著,用接合的DNA來改變八cW XLl-Blue細胞,而 含有活性酵素的無性繁殖個體則用在活的有機體中的分析 來選擇,選擇幾個無性繁殖個體並加以排序,以確定如第 14圖所示的排序之合成luc基因的存在與精確性,完整的 〇RF則稱爲IDRIS (FA)。 將合成的基因接種到在聚合鏈中(polylinker)位於BamH I與Sal I之間的載體pBSK(+)中,在此位置中基因不會在具 有alpha膊的架構中,且與lacy促進劑具有一段明顯的距 離。無論如何,會產生足夠的螢光素,使其可以展現酵素 的基本特性,使用Promega lysis方法來自培養整夜的微生 物中製備含有IDRIS (FA)的五⑺//Xll-Blue細胞之天然細胞 任意的萃取物。 接著,在50°C下測試萃取物中的酵素的熱穩定性超過 20分鐘,且與熱穩定的變異物E354I+D357Y比較,相較於 變異物E354I+D357Y,運用三變異物的新密碼單元明顯的 具有更好的熱穩定性(見第15圖)。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).
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