JP2014001161A - Calcium sensor protein using red fluorescent protein - Google Patents
Calcium sensor protein using red fluorescent protein Download PDFInfo
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- JP2014001161A JP2014001161A JP2012137434A JP2012137434A JP2014001161A JP 2014001161 A JP2014001161 A JP 2014001161A JP 2012137434 A JP2012137434 A JP 2012137434A JP 2012137434 A JP2012137434 A JP 2012137434A JP 2014001161 A JP2014001161 A JP 2014001161A
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Abstract
Description
本発明は、特定部位のアミノ酸を置換した赤色蛍光蛋白質（以下、ＲＦＰ）、又はそのホモログを用いたカルシウムセンサー蛋白質に関する。より具体的には、従来の赤色蛍光カルシウムセンサー蛋白質よりも更に反応性に優れた、かつ細胞内で発現後に細胞質から核内への移行を示さない、カルシウムセンサー蛋白質、及び前記カルシウムセンサー蛋白質をコードするカルシウムセンサー遺伝子に関する。 The present invention relates to a calcium sensor protein using a red fluorescent protein (hereinafter referred to as RFP) in which an amino acid at a specific site is substituted, or a homologue thereof. More specifically, the calcium sensor protein, which is more reactive than the conventional red fluorescent calcium sensor protein and does not show a transition from the cytoplasm to the nucleus after expression in the cell, and the calcium sensor protein is encoded. It relates to the calcium sensor gene.
カルシウムは生体にとって、構造の維持に必須である骨の主要な構成成分であると同時に、筋肉の収縮、神経興奮性やホルモン分泌、酵素活性の変化などの各種の細胞機能の調節因子として、生体機能の維持および調節に不可欠な役割を担っている。このため、生体内（細胞外及び細胞内）のカルシウム変動を探知し、カルシウム濃度を測定するのに用いられるカルシウムセンサーの重要性が高まっている。
カルシウムセンサーは大きく分けて4種類のものがこれまでに開発されている。以下にその概要を示す。
Calcium is a major component of bone that is essential for the maintenance of the structure for the living body, and at the same time as a regulator of various cellular functions such as muscle contraction, nerve excitability, hormone secretion, and changes in enzyme activity. It plays an essential role in maintaining and regulating function. For this reason, the importance of the calcium sensor used for detecting calcium fluctuation in the living body (extracellular and intracellular) and measuring the calcium concentration is increasing.
Four types of calcium sensors have been developed so far. The outline is shown below.
１）カルシウム感受性の合成色素：カルシウムに感受性のある化学合成された色素であり、現在一般によく使用されている。細胞内において使用する場合は、外部から細胞に取り込ませる必要があるが、特定の細胞のみに色素を取り込ませることは難しく、ガラス針等により細胞に該色素を注入しなければならないという問題点を有する。 1) Calcium-sensitive synthetic pigments: These are chemically synthesized pigments sensitive to calcium, and are currently commonly used. When used in a cell, it is necessary to make the cell take in from outside, but it is difficult to make the dye take in only a specific cell, and the dye must be injected into the cell with a glass needle or the like. Have.
２）エクオリン：カルシウムに反応して発光する蛋白質であり、細胞に直接注入するか、該蛋白質を産生する遺伝子を細胞に導入して使用する。細胞内で機能するためには細胞に補酵素を供給する必要があり、また発光が極めて微弱であるという問題点を有する。 2) Aequorin: a protein that emits light in response to calcium, and is directly injected into a cell, or a gene that produces the protein is introduced into a cell and used. In order to function in a cell, it is necessary to supply a coenzyme to the cell, and there is a problem that luminescence is extremely weak.
３）蛍光共鳴エネルギー移動（ＦＲＥＴ）を応用したカルシウム感受性蛋白質：カルシウムに感受性のあるカルモジュリン（ＣａＭ）とそれに結合するミオシン軽鎖キナーゼの一部の配列、二つの色の異なるＧＦＰ又はそのホモログを結合した蛋白質であり、カルシウムがＣａＭに結合するとその構造が変化し、ＦＲＥＴを起こして二つのＧＦＰまたはそのホモログの発する蛍光強度が変化することを利用している。該蛋白質は、細胞に直接注入するか、該蛋白質をコードする遺伝子を細胞に取り込ませて使用する。ＦＲＥＴにおる蛍光変化は軽微であり、さらに一般的に用いられているアルゴンレーザーを搭載したレーザー顕微鏡により測定することが出来ないという問題点がある。 3) Calcium-sensitive protein using fluorescence resonance energy transfer (FRET): Calcium-sensitive calmodulin (CaM) and a partial sequence of myosin light chain kinase that binds to it, two different colors of GFP or their homologs This protein utilizes the fact that when calcium binds to CaM, its structure changes, and FRET occurs to change the fluorescence intensity emitted by two GFPs or their homologues. The protein is used by directly injecting into a cell or by allowing a gene encoding the protein to be taken into the cell. There is a problem that the fluorescence change in FRET is slight and cannot be measured by a laser microscope equipped with a commonly used argon laser.
４）一つのＧＦＰ又はＲＦＰからなるカルシウム感受性蛋白質：ＧＦＰ又はＲＦＰ、好ましくはｍＡｐｐｌｅ、の改変体にＣａＭとミオシン軽鎖キナーゼの一部の配列を結合したカルシウム感受性蛋白質であり、カルシウムがＣａＭに結合すると蛋白質の構造が変化し、ＧＦＰ又はＲＦＰの発する蛍光強度が変化することを利用している。該蛋白質も、細胞に直接注入するか、その他遺伝子を細胞に取り込ませて使用する。一般にカルシウムに対する感度が低く、実際の細胞では信号／雑音比が低いため、測定が困難であるという問題点を有する。また従来のｍＡｐｐｌｅからなるカルシウム感受性蛋白質（R-GECO1：非特許文献１）は細胞内での発現部位が細胞質と核内であるため、該蛋白質を用いた測定では同一細胞の細胞質と核において異なるタイミングで蛍光変化が生じる場合があるという問題点がある。 4) A calcium-sensitive protein consisting of one GFP or RFP: a calcium-sensitive protein in which a partial sequence of CaM and myosin light chain kinase is bound to a variant of GFP or RFP, preferably mAapple, and calcium binds to CaM. This utilizes the fact that the protein structure changes and the fluorescence intensity emitted by GFP or RFP changes. The protein is also used by directly injecting it into cells or by incorporating other genes into cells. In general, since the sensitivity to calcium is low and the signal / noise ratio is low in an actual cell, there is a problem that measurement is difficult. In addition, since the calcium-sensitive protein (R-GECO1: Non-patent document 1) made of conventional apple is expressed in the cytoplasm and nucleus in the cell, the measurement using the protein differs in the cytoplasm and nucleus of the same cell. There is a problem that fluorescence changes may occur at the timing.
本発明者らは、上記４）の応用として、ＧＦＰの蛍光特性を制御することが可能なカルシウムセンサー蛋白質を作成する方法、並びに該方法により作成されるカルシウムセンサー蛋白質、および該カルシウムセンサー蛋白質をコードするカルシウムセンサー遺伝子を提供し（特許文献１）、カルシウムに対する感度が従来のカルシウムセンサーに比して高く、かつ特定細胞への取り込みが容易であり、更に測定に特別な装置及び補酵素等を必要としないカルシウムセンサー蛋白質の作成に成功している（特許文献２、特許文献３）。
しかし、近年、生体内でのカルシウムの微少な変動を感知する必要性が以前にも増して高まっており、上記特許文献１、特許文献２及び特許文献３のカルシウムセンサー蛋白質をもってしても、十分な成果が上げられない状況となっている。
As an application of the above 4), the present inventors have prepared a method for producing a calcium sensor protein capable of controlling the fluorescence characteristics of GFP, a calcium sensor protein produced by the method, and a code for the calcium sensor protein. Provides a calcium sensor gene that can be used (Patent Document 1), has higher sensitivity to calcium than conventional calcium sensors, is easily incorporated into specific cells, and requires special equipment and coenzymes for measurement. Have succeeded in producing a calcium sensor protein (Patent Document 2, Patent Document 3).
However, in recent years, there has been an ever-increasing need to detect minute fluctuations of calcium in a living body, and even with the calcium sensor proteins of Patent Document 1, Patent Document 2, and Patent Document 3, sufficient It is a situation that can not be achieved.
また近年、光刺激で細胞機能を操作し、同時に蛍光カルシウムイメージングで細胞機能を測定するという実験の要求が高まってきた。蛍光カルシウムイメージングに応用される赤色蛍光カルシウムセンサー蛋白質は、その励起波長が細胞機能操作を目的として汎用される光刺激プローブＣｈａｎｎｅｌｒｈｏｄｏｐｓｉｎ−２（非特許文献２）の活性化波長と重複しないため、Ｃｈａｎｎｅｌｒｈｏｄｏｐｓｉｎ−２との併用が可能である。つまり赤色蛍光カルシウムセンサー蛋白質によって細胞機能操作と細胞機能測定を同時に行う実験が可能となる。そのためＣｈａｎｎｅｌｒｈｏｄｏｐｓｉｎ−２と併用可能な赤色蛍光カルシウムセンサー蛋白質の開発が強く望まれている状況である。 In recent years, there has been a growing demand for experiments in which cell functions are manipulated by light stimulation and at the same time cell functions are measured by fluorescent calcium imaging. The red fluorescent calcium sensor protein applied to fluorescent calcium imaging has an excitation wavelength that does not overlap with the activation wavelength of the light stimulating probe Channelrhodopsin-2 (Non-patent Document 2), which is widely used for the purpose of cell function manipulation. 2 can be used together. In other words, the red fluorescent calcium sensor protein enables an experiment in which cell function manipulation and cell function measurement are performed simultaneously. For this reason, development of a red fluorescent calcium sensor protein that can be used in combination with Channelrhodopsin-2 is strongly desired.
上記事情に鑑み、本発明は、従来の赤色蛍光カルシウムセンサーよりも、さらに、反応性に優れ、かつ細胞内で発現後に細胞質から核内への移行を示さない、カルシウムセンサー蛋白質、及び、該蛋白質をコードする遺伝子の提供を目的とする。 In view of the above circumstances, the present invention provides a calcium sensor protein, which is more reactive than a conventional red fluorescent calcium sensor and does not show a transition from cytoplasm to nucleus after expression in the cell, and the protein It aims at providing the gene which codes.
発明者らは、既存の赤色蛍光カルシウムセンサーであるＲ−ＧＥＣＯ１よりも高い蛍光反応性を示すセンサーの開発を目的として鋭意研究を行ったところ、まず、Ｒ−ＧＥＣＯ１よりも約１．４倍の高い蛍光反応性を示すセンター、Ｒ−ＣａＭＰ１．０１を完成させた。しかし、Ｒ−ＣａＭＰ１．０１は、Ｒ−ＧＥＣＯ１と同様に発現後に核内にも局在するものであった。細胞質カルシウム測定を目的とした研究において、この局在パターンは好ましいものではない。その理由としては、１）細胞質と核内ではカルシウム濃度変化に時差があり（非特許文献３）、細胞質と核での蛍光シグナルを分離して検出する必要がある、２）カルシウムセンサーの分解産物が核内に局在した細胞では通常の細胞質カルシウム応答パターンに異変が見られたという報告（非特許文献４）から、核内局在による細胞機能への悪影響が考えられる、等が挙げられる。
そこで、発明者らは、カルシウムセンサーとしての性能はセンサーのＮ末端やＣ末端へのペプチド付加によって修飾を受けるとの知見に基づき（非特許文献５）、Ｒ−ＣａＭＰ１．０１の更なる改良を目的として、Ｒ−ＣａＭＰ１．０１へ種々様々なペプチドを付加した改変体を作製し、その機能を検討した。その結果、Ｒ−ＣａＭＰ１．０１のＣ末端に１５アミノ酸のリンカーと２２アミノ酸からなるペプチド（配列番号１２）を付加した改変体（以下、Ｒ−ＣａＭＰ１．０７と称する）は、核内への局在を示さず、かつＲ−ＣａＭＰ１．０１よりもさらに高い蛍光反応性を示すことを見出した。
本発明は以上の知見に基づいて完成されたものである。
The inventors conducted intensive research for the purpose of developing a sensor exhibiting higher fluorescence reactivity than R-GECO1, which is an existing red fluorescent calcium sensor. First, about 1.4 times that of R-GECO1. A center exhibiting high fluorescence reactivity, R-CaMP1.01, was completed. However, R-CaMP1.01 was also localized in the nucleus after expression in the same manner as R-GECO1. In studies aimed at measuring cytosolic calcium, this localization pattern is not preferred. The reasons are as follows: 1) There is a time difference in changes in calcium concentration in the cytoplasm and nucleus (Non-patent Document 3), and it is necessary to separate and detect the fluorescence signals in the cytoplasm and nucleus. 2) Degradation products of the calcium sensor From the report (Non-patent Document 4) that abnormal changes were observed in the normal cytoplasmic calcium response pattern in cells localized in the nucleus, there is an adverse effect on the cell function due to the localization in the nucleus.
Therefore, the inventors have further improved R-CaMP1.01 based on the knowledge that the performance as a calcium sensor is modified by adding a peptide to the N-terminus or C-terminus of the sensor (Non-Patent Document 5). For the purpose, the modified body which added various peptides to R-CaMP1.01 was produced, and the function was examined. As a result, a modified form (hereinafter referred to as R-CaMP1.07) in which a 15-amino acid linker and a 22-amino acid peptide (SEQ ID NO: 12) were added to the C-terminus of R-CaMP1.01 was It was found that the fluorescent reactivity was higher than that of R-CaMP1.01.
The present invention has been completed based on the above findings.
すなわち、本発明は以下の（１）〜（１０）に関する。
（１）下記（ａ）〜（ｋ）のアミノ酸配列を、Ｎ末端から順に有することを特徴とするカルシウムセンサー蛋白質：
（ａ）配列番号１で示されるアミノ酸配列；
（ｂ）３つのアミノ酸からなる配列 Ｍｅｔ−Ｘａａ１−Ｘａａ２（ここでＸａａ１及びＸａａ２はそれぞれ独立して任意のアミノ酸である）（リンカーＡ）（配列番号２）；
（ｃ）ミオシン軽鎖キナーゼ蛋白質、又はカルモジュリン結合部位を含むその部分アミノ酸配列；
（ｄ）前記（ｃ）の配列と後記（ｅ）の配列とを連結する、Ｉｓｏ−Ｉｓｏ、Ｉｓｏ−Ｌｅｕ、Ｉｓｏ−Ｍｅｔ、Ｉｓｏ−Ｐｒｏ、Ｉｓｏ−Ｖａｌ、Ｉｓｏ−Ｇｌｙ、Ｉｓｏ−Ａｌａ、Ｌｅｕ−Ｉｓｏ、Ｌｅｕ−Ｌｅｕ、Ｌｅｕ−Ｍｅｔ、Ｌｅｕ−Ｐｒｏ、Ｌｅｕ−Ｖａｌ、Ｌｅｕ−Ｇｌｙ、Ｌｅｕ−Ａｌａ、Ｍｅｔ−Ｉｓｏ、Ｍｅｔ−Ｌｅｕ、Ｍｅｔ−Ｍｅｔ、Ｍｅｔ−Ｐｒｏ、Ｍｅｔ−Ｖａｌ、Ｍｅｔ−Ｇｌｙ、Ｍｅｔ−Ａｌａ、Ｐｒｏ−Ｉｓｏ、Ｐｒｏ−Ｌｅｕ、Ｐｒｏ−Ｍｅｔ、Ｐｒｏ−Ｐｒｏ、Ｐｒｏ−Ｖａｌ、Ｐｒｏ−Ｇｌｙ、Ｐｒｏ−Ａｌａ、Ｖａｌ−Ｉｓｏ、Ｖａｌ−Ｌｅｕ、Ｖａｌ−Ｍｅｔ、Ｖａｌ−Ｐｒｏ、Ｖａｌ−Ｖａｌ、Ｖａｌ−Ｇｌｙ、Ｖａｌ−Ａｌａ、Ｇｌｙ−Ｉｓｏ、Ｇｌｙ−Ｌｅｕ、Ｇｌｙ−Ｍｅｔ、Ｇｌｙ−Ｐｒｏ、Ｇｌｙ−Ｖａｌ、Ｇｌｙ−Ｇｌｙ、Ｇｌｙ−Ａｌａ、Ａｌａ−Ｉｓｏ、Ａｌａ−Ｌｅｕ、Ａｌａ−Ｍｅｔ、Ａｌａ−Ｐｒｏ、Ａｌａ−Ｖａｌ、Ａｌａ−Ｇｌｙ及びＡｌａ−Ａｌａからなる群より選択される何れか一のアミノ酸配列（リンカーＢ）；
（ｅ）配列番号３で示される配列のＸ番目〜２３６番目までのアミノ酸配列であって、１５１番目及び／又は１５２番目及び／又は１６９番目及び／又は１７１番目及び／又は２１９番目のアミノ酸を他のアミノ酸に置換したもの（ここで、Ｘは１５１〜１５３の任意の位置である）；
（ｆ）前記（ｅ）の配列と後記（ｇ）の配列を連結する、６つのアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｘａａ５−Ｇｌｙ−Ｇｌｙ−Ｘａａ６（ここでＸａａ５及びＸａａ６はそれぞれ独立して任意のアミノ酸である）（配列番号４）；
（ｇ）配列番号３で示される配列の１番目〜Ｙ番目までのアミノ酸配列であって、
１番目及び／又は８番目及び／又は５２番目及び／又は５４番目及び／又は７６番目及び／又は１３６番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列（ここで、Ｙは１４４〜１５０の任意の位置である）；
（ｈ）前記（ｇ）の配列と後記（ｉ）の配列とを連結するアミノ酸配列Ｔｈｒ−Ａｒｇ、Ｐｈｅ−Ａｒｇ、Ｔｒｐ−Ａｒｇ、Ｔｙｒ−Ａｒｇ、Ｇｌｙ−Ａｒｇ、Ａｌａ−Ａｒｇ又はＴｈｒ（リンカーＣ）；
（ｉ）配列番号９で示される配列の２番目〜１４８番目までのアミノ酸配列であって、６３番目及び／又は７７番目及び／又は１０１番目及び／又は１１１番目及び／又は１２７番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列；
（ｊ）前記（ｉ）の配列と後記（ｋ）の配列とを連結する、１５個のアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ７−Ｇｌｙ−Ｇｌｙ−Ｘａａ８−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ９−Ｘａａ１０（ここでＸａａ７、Ｘａａ８、Ｘａａ９及びＸａａ１０はそれぞれ独立して任意のアミノ酸である）（リンカーＤ）（配列番号１０）；
（ｋ）Ｐｒｏ−Ｖａｌ−Ｌｙｓ−Ｇｌｎ−Ｔｈｒ−Ｌｅｕ−Ａｓｎ−Ｐｈｅ−Ａｓｐ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１２）、Ｇｌｎ−Ｃｙｓ−Ｔｈｒ−Ａｓｎ−Ｔｙｒ−Ａｌａ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１３）、Ｇｌｕ−Ｇｌｙ−Ａｒｇ−Ｇｌｙ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｔｈｒ−Ｃｙｓ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１４）又はＡｌａ−Ｔｈｒ−Ａｓｎ−Ｐｈｅ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｇｌｎ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１５）のいずれかのアミノ酸配列。
That is, the present invention relates to the following (1) to (10).
(1) Calcium sensor protein having the following amino acid sequences (a) to (k) in order from the N-terminus:
(A) the amino acid sequence represented by SEQ ID NO: 1;
(B) a sequence consisting of three amino acids Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each independently any amino acid) (linker A) (SEQ ID NO: 2);
(C) a myosin light chain kinase protein, or a partial amino acid sequence thereof comprising a calmodulin binding site;
(D) The sequence of (c) and the sequence of (e) described below are linked, Iso-Iso, Iso-Leu, Iso-Met, Iso-Pro, Iso-Val, Iso-Gly, Iso-Ala, Leu -Iso, Leu-Leu, Leu-Met, Leu-Pro, Leu-Val, Leu-Gly, Leu-Ala, Met-Iso, Met-Leu, Met-Met, Met-Pro, Met-Val, Met-Gly , Met-Ala, Pro-Iso, Pro-Leu, Pro-Met, Pro-Pro, Pro-Val, Pro-Gly, Pro-Ala, Val-Iso, Val-Leu, Val-Met, Val-Pro, Val -Val, Val-Gly, Val-Ala, Gly-Iso, Gly-Leu, Gly-Met, Gl Any selected from the group consisting of Pro, Gly-Val, Gly-Gly, Gly-Ala, Ala-Iso, Ala-Leu, Ala-Met, Ala-Pro, Ala-Val, Ala-Gly and Ala-Ala Any one amino acid sequence (linker B);
(E) the amino acid sequence from the Xth to the 236th amino acid sequence of the sequence shown in SEQ ID NO: 3, except for the 151st and / or 152th and / or 169th and / or 171st and / or 219th amino acids (Wherein X is an arbitrary position of 151 to 153);
(F) A sequence Gly-Gly-Xaa5-Gly-Gly-Xaa6 consisting of six amino acid sequences connecting the sequence of (e) and the sequence of (g) to be described later (where Xaa5 and Xaa6 are each independently arbitrary (SEQ ID NO: 4);
(G) an amino acid sequence from the first to the Y-th of the sequence represented by SEQ ID NO: 3,
Amino acid sequence in which the first and / or eighth and / or 52 and / or 54 and / or 76 and / or 136 amino acids are substituted with other amino acids (where Y is any of 144 to 150) Position);
(H) Amino acid sequence Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg or Thr (linker C) linking the sequence of (g) and the sequence of (i) described later );
(I) Amino acid sequence from 2nd to 148th of the sequence shown in SEQ ID NO: 9, except for the 63rd and / or 77th and / or 101st and / or 111th and / or 127th amino acids An amino acid sequence substituted with the amino acid of
(J) A sequence consisting of 15 amino acid sequences Gly-Gly-Gly-Xaa7-Gly-Gly-Xaa8-Gly-Gly-Gly-Gly, linking the sequence of (i) and the sequence of (k) described later -Gly-Gly-Xaa9-Xaa10 (where Xaa7, Xaa8, Xaa9 and Xaa10 are each independently any amino acid) (linker D) (SEQ ID NO: 10);
(K) Pro-Val-Lys-Gln-Thr-Leu-Asn-Phe-Asp-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 12 ), Gln-Cys-Thr-Asn-Tyr-Ala-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 13), Glu-Gly-Arg -Gly-Ser-Leu-Leu-Thr-Cys-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly (SEQ ID NO: 14) or Ala-Thr-Asn-Phe-Ser-Leu-Leu-Lys- Gln-Ala-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly Any of the amino acid sequences of the number 15).
（２）前記（ａ）の配列の５番目から１０番目に存在するＨｉｓの数が０〜５であり、及び／又は前記（ａ）の配列の２番目のＡｒｇが欠失し、及び／又は前記（ｅ）の配列の１５１番目のアミノ酸がＩｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｔｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｅ）の配列の１５２番目のアミノ酸がＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｓｅｒ、Ａｓｐ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｅ）の配列の１６９番目のアミノ酸がＨｉｓ、Ｌｙｓ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｅ）の配列の１７１番目のアミノ酸がＡｒｇ、Ｈｉｓ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｅ）の配列の２１９番目のアミノ酸がＰｈｅ、Ｔｒｐ、Ｔｈｒ、Ｓｅｒ、Ｃｙｓ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の１番目のアミノ酸がＩｓｏ、Ｌｅｕ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の８番目のアミノ酸がＧｌｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の５２番目のアミノ酸がＡｒｇ、Ｈｉｓ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の５４番目のアミノ酸がＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｓｅｒ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の７６番目のアミノ酸がＩｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｇ）の配列の１３６番目のアミノ酸がＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｉ）の配列において、配列番号９で示される配列の６３番目のアミノ酸がＬｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｔｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｓｅｒ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｉ）の配列において、配列番号９で示される配列の７７番目のアミノ酸がＡｒｇ、Ｈｉｓ、Ａｓｎ、Ｇｌｎ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｉ）の配列において、配列番号９で示される配列の１０１番目のアミノ酸がＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｉ）の配列において、配列番号９で示される配列の１１１番目のアミノ酸がＧｌｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｙ若しくはＡｌａに置換され、及び／又は前記（ｉ）の配列において、配列番号９で示される配列の１２７番目のアミノ酸がＡｓｐ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａに置換されることを特徴とする上記（１）に記載のカルシウムセンサー蛋白質。 (2) The number of His present in the 5th to 10th positions of the sequence (a) is 0 to 5 and / or the second Arg of the sequence (a) is deleted, and / or The 151st amino acid of the sequence (e) is substituted with Iso, Leu, Met, Pro, Val, Tyr, Phe, Trp, Thr, Gly or Ala, and / or the 152nd amino acid of the sequence (e) An amino acid is substituted with Tyr, Phe, Trp, Thr, Ser, Asp, Gly or Ala, and / or the 169th amino acid of the sequence of (e) is substituted with His, Lys, Gly or Ala, and / or The 171st amino acid of the sequence (e) is substituted with Arg, His, Gly or Ala, and / or the 219th amino acid of the sequence (e) is Phe. Substituted with Trp, Thr, Ser, Cys, Gly or Ala and / or the first amino acid of the sequence (g) is substituted with Iso, Leu, Pro, Val, Gly or Ala and / or g) the 8th amino acid in the sequence of Gln, Asp, Glu, Gly or Ala, and / or the 52nd amino acid of the sequence of (g) is Arg, His, Iso, Leu, Met, Pro, Substituted with Val, Gly or Ala, and / or the 54th amino acid of the sequence of (g) is substituted with Tyr, Phe, Trp, Ser, Iso, Leu, Met, Pro, Val, Gly or Ala; and / Or the 76th amino acid of the sequence (g) is Iso, Leu, Met, Pro, Gly or Ala And / or the 136th amino acid of the sequence (g) is replaced with Tyr, Phe, Trp, Thr, Iso, Leu, Met, Pro, Val, Gly or Ala, and / or (i) In the sequence of SEQ ID NO: 9, the 63rd amino acid of the sequence represented by SEQ ID NO: 9 is substituted with Leu, Met, Pro, Val, Tyr, Phe, Trp, Thr, Ser, Gly or Ala, and / or the above (i) In the sequence, the 77th amino acid of the sequence represented by SEQ ID NO: 9 is substituted with Arg, His, Asn, Gln, Gly or Ala, and / or in the sequence of (i), the sequence represented by SEQ ID NO: 9 101st amino acid is substituted with Tyr, Phe, Trp, Thr, Gly or Ala, and / or the above (i) In the sequence, the 111th amino acid of the sequence represented by SEQ ID NO: 9 is substituted with Gln, Asp, Glu, Gly or Ala, and / or the 127th sequence of the sequence represented by SEQ ID NO: 9 in the sequence (i) The calcium sensor protein according to (1) above, wherein the amino acid is substituted with Asp, Iso, Leu, Met, Pro, Val, Gly or Ala.
（３）前記（ｅ）の配列が配列番号３で示される配列の１５１番目〜２３６番目までのアミノ酸配列であり、前記（ｇ）の配列が配列番号３で示される配列の１番目〜１５０番目までのアミノ酸配列であることを特徴とする上記（１）又は（２）に記載のカルシウムセンサー蛋白質。 (3) The sequence of (e) is the amino acid sequence 151 to 236 of the sequence represented by SEQ ID NO: 3, and the sequence of (g) is the first to 150th of the sequence represented by SEQ ID NO: 3. The calcium sensor protein according to (1) or (2) above, which has an amino acid sequence up to
（４）前記（ｂ）の配列が配列番号５若しくは配列番号６で示されるアミノ酸配列であり、及び／又は前記（ｃ）の配列が配列番号７で示されるアミノ酸配列であり、及び／又は前記（ｆ）の配列が配列番号８で示されるアミノ酸配列であることを特徴とする上記（１）乃至（３）のいずれかに記載のカルシウムセンサー蛋白質。 (4) The sequence of (b) is an amino acid sequence represented by SEQ ID NO: 5 or 6 and / or the sequence of (c) is an amino acid sequence represented by SEQ ID NO: 7, and / or The calcium sensor protein according to any one of (1) to (3) above, wherein the sequence (f) is the amino acid sequence represented by SEQ ID NO: 8.
（５）前記（ｂ）の配列が配列番号６で示されるアミノ酸配列であり、前記（ｄ）の配列がＰｒｏ−Ｖａｌであり、前記（ｈ）の配列がＴｈｒ−Ａｒｇであることを特徴とする上記（４）に記載のカルシウムセンサー蛋白質。 (5) The sequence of (b) is an amino acid sequence represented by SEQ ID NO: 6, the sequence of (d) is Pro-Val, and the sequence of (h) is Thr-Arg, The calcium sensor protein according to (4) above.
（６）前記（ｅ）の配列がアミノ酸の置換を含まない配列番号３で示される配列の１５１番目〜２３６番目までのアミノ酸配列であり、前記（ｇ）の配列がアミノ酸の置換を含まない配列番号３で示される配列の１番目〜１５０番目であり、前記（ｉ）の配列がアミノ酸の置換を含まない配列番号９で示される配列の２番目〜１４８番目であることを特徴とする上記（５）に記載のカルシウムセンサー蛋白質。 (6) The sequence of (e) is an amino acid sequence from position 151 to position 236 of the sequence represented by SEQ ID NO: 3 that does not include amino acid substitution, and the sequence of (g) does not include amino acid substitution (1) to the 150th sequence of the sequence represented by No. 3, and the sequence (i) is the second to 148th sequence of the sequence represented by SEQ ID No. 9 that does not include amino acid substitution ( The calcium sensor protein according to 5).
（７）前記（ｅ）の配列が配列番号３で示される配列の１５１番目〜２３６番目であって、１５１番目のアミノ酸がＶａｌに置換され、１５２番目のアミノ酸がＳｅｒに置換され、１６９番目のアミノ酸がＧｌｙに置換され、１７１番目のアミノ酸がＡｒｇに置換され、２１９番目のアミノ酸がＣｙｓに置換され、前記（ｇ）の配列が配列番号３で示される配列の１番目〜１５０番目までのアミノ酸配列であり、１番目のアミノ酸がＬｅｕに置換され、８番目のアミノ酸がＡｓｐに置換され、５２番目のアミノ酸がＶａｌに置換され、５４番目のアミノ酸がＶａｌに置換され、７６番目のアミノ酸がＡｌａに置換され、１３６番目のアミノ酸がＰｒｏに置換され、前記（ｉ）の配列が配列番号９で示される配列の６３番目のアミノ酸がＰｈｅに置換され、７７番目のアミノ酸がＡｓｎに置換され、１０１番目のアミノ酸がＧｌｙに置換され、１１１番目のアミノ酸がＡｓｐに置換され、１２７番目のアミノ酸がＶａｌに置換されていることを特徴とする上記（５）に記載のカルシウムセンサー蛋白質。 (7) The sequence of (e) is from position 151 to position 236 of the sequence represented by SEQ ID NO: 3, wherein the 151st amino acid is replaced with Val, the 152nd amino acid is replaced with Ser, and the 169th Amino acid is substituted with Gly, 171st amino acid is substituted with Arg, 219th amino acid is substituted with Cys, and the sequence of (g) is the first to 150th amino acids of the sequence represented by SEQ ID NO: 3 The first amino acid is replaced with Leu, the eighth amino acid is replaced with Asp, the 52nd amino acid is replaced with Val, the 54th amino acid is replaced with Val, and the 76th amino acid is replaced with Ala. And the 136th amino acid is substituted with Pro, and the 63rd amino acid of the sequence represented by SEQ ID NO: 9 is the Phe Wherein the 77th amino acid is substituted with Asn, the 101st amino acid is substituted with Gly, the 111th amino acid is substituted with Asp, and the 127th amino acid is substituted with Val. The calcium sensor protein according to (5).
（８）前記（ｊ）の配列が配列番号１０で示される配列であることを特徴とする上記（１）乃至（７）のいずれかに記載のカルシウムセンサー蛋白質。 (8) The calcium sensor protein according to any one of (1) to (7) above, wherein the sequence of (j) is the sequence represented by SEQ ID NO: 10.
（９）前記（ｋ）の配列が配列番号１２で示される配列であることを特徴とする上記（１）乃至（８）のいずれかに記載のカルシウムセンサー蛋白質。 (9) The calcium sensor protein according to any one of (1) to (8) above, wherein the sequence of (k) is the sequence represented by SEQ ID NO: 12.
（１０）上記（１）乃至（９）のいずれかに記載の蛋白質をコードするカルシウムセンサー遺伝子。 (10) A calcium sensor gene encoding the protein according to any one of (1) to (9) above.
本発明により、従来の赤色蛍光カルシウムセンサーに比べ、更に反応性に優れ、かつ細胞内で発現後に細胞質から核内への移行を示さない、カルシウムセンサー蛋白質の提供が可能となる。 According to the present invention, it is possible to provide a calcium sensor protein that is more reactive than a conventional red fluorescent calcium sensor and that does not show a transition from cytoplasm to nucleus after expression in cells.
本発明のカルシウムセンサー蛋白質は、下記（ａ）〜（ｋ）のアミノ酸配列を、Ｎ末端から順に有することを特徴とする蛋白質である：
（ａ）配列番号１で示されるアミノ酸配列；
（ｂ）３つのアミノ酸からなる配列 Ｍｅｔ−Ｘａａ１−Ｘａａ２（ここでＸａａ１及びＸａａ２はそれぞれ独立して任意のアミノ酸である）（リンカーＡ）（配列番号２）；
（ｃ）ミオシン軽鎖キナーゼ蛋白質、又はカルモジュリン結合部位を含むその部分アミノ酸配列；
（ｄ）前記（ｃ）の配列と後記（ｅ）の配列とを連結する、Ｉｓｏ−Ｉｓｏ、Ｉｓｏ−Ｌｅｕ、Ｉｓｏ−Ｍｅｔ、Ｉｓｏ−Ｐｒｏ、Ｉｓｏ−Ｖａｌ、Ｉｓｏ−Ｇｌｙ、Ｉｓｏ−Ａｌａ、Ｌｅｕ−Ｉｓｏ、Ｌｅｕ−Ｌｅｕ、Ｌｅｕ−Ｍｅｔ、Ｌｅｕ−Ｐｒｏ、Ｌｅｕ−Ｖａｌ、Ｌｅｕ−Ｇｌｙ、Ｌｅｕ−Ａｌａ、Ｍｅｔ−Ｉｓｏ、Ｍｅｔ−Ｌｅｕ、Ｍｅｔ−Ｍｅｔ、Ｍｅｔ−Ｐｒｏ、Ｍｅｔ−Ｖａｌ、Ｍｅｔ−Ｇｌｙ、Ｍｅｔ−Ａｌａ、Ｐｒｏ−Ｉｓｏ、Ｐｒｏ−Ｌｅｕ、Ｐｒｏ−Ｍｅｔ、Ｐｒｏ−Ｐｒｏ、Ｐｒｏ−Ｖａｌ、Ｐｒｏ−Ｇｌｙ、Ｐｒｏ−Ａｌａ、Ｖａｌ−Ｉｓｏ、Ｖａｌ−Ｌｅｕ、Ｖａｌ−Ｍｅｔ、Ｖａｌ−Ｐｒｏ、Ｖａｌ−Ｖａｌ、Ｖａｌ−Ｇｌｙ、Ｖａｌ−Ａｌａ、Ｇｌｙ−Ｉｓｏ、Ｇｌｙ−Ｌｅｕ、Ｇｌｙ−Ｍｅｔ、Ｇｌｙ−Ｐｒｏ、Ｇｌｙ−Ｖａｌ、Ｇｌｙ−Ｇｌｙ、Ｇｌｙ−Ａｌａ、Ａｌａ−Ｉｓｏ、Ａｌａ−Ｌｅｕ、Ａｌａ−Ｍｅｔ、Ａｌａ−Ｐｒｏ、Ａｌａ−Ｖａｌ、Ａｌａ−Ｇｌｙ及びＡｌａ−Ａｌａからなる群より選択される何れか一のアミノ酸配列（リンカーＢ）；
（ｅ）配列番号３で示される配列のＸ番目〜２３６番目までのアミノ酸配列であって、１５１番目及び／又は１５２番目及び／又は１６９番目及び／又は１７１番目及び／又は２１９番目のアミノ酸を他のアミノ酸に置換したもの（ここで、Ｘは１５１〜１５３の任意の位置である）；
（ｆ）前記（ｅ）の配列と後記（ｇ）の配列を連結する、６つのアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｘａａ５−Ｇｌｙ−Ｇｌｙ−Ｘａａ６（ここでＸａａ５及びＸａａ６はそれぞれ独立して任意のアミノ酸である）（配列番号４）；
（ｇ）配列番号３で示される配列の１番目〜Ｙ番目までのアミノ酸配列であって、
１番目及び／又は８番目及び／又は５２番目及び／又は５４番目及び／又は７６番目及び／又は１３６番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列（ここで、Ｙは１４４〜１５０の任意の位置である）；
（ｈ）前記（ｇ）の配列と後記（ｉ）の配列とを連結するアミノ酸配列Ｔｈｒ−Ａｒｇ、Ｐｈｅ−Ａｒｇ、Ｔｒｐ−Ａｒｇ、Ｔｙｒ−Ａｒｇ、Ｇｌｙ−Ａｒｇ、Ａｌａ−Ａｒｇ又はＴｈｒ（リンカーＣ）；
（ｉ）配列番号９で示される配列の２番目〜１４８番目までのアミノ酸配列であって、６３番目及び／又は７７番目及び／又は１０１番目及び／又は１１１番目及び／又は１２７番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列；
（ｊ）前記（ｉ）の配列と後記（ｋ）の配列とを連結する、１５個のアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ７−Ｇｌｙ−Ｇｌｙ−Ｘａａ８−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ９−Ｘａａ１０（ここでＸａａ７、Ｘａａ８、Ｘａａ９及びＸａａ１０はそれぞれ独立して任意のアミノ酸である）（リンカーＤ）（配列番号１０）；
（ｋ）Ｐｒｏ−Ｖａｌ−Ｌｙｓ−Ｇｌｎ−Ｔｈｒ−Ｌｅｕ−Ａｓｎ−Ｐｈｅ−Ａｓｐ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１２）、Ｇｌｎ−Ｃｙｓ−Ｔｈｒ−Ａｓｎ−Ｔｙｒ−Ａｌａ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１３）、Ｇｌｕ−Ｇｌｙ−Ａｒｇ−Ｇｌｙ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｔｈｒ−Ｃｙｓ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１４）又はＡｌａ−Ｔｈｒ−Ａｓｎ−Ｐｈｅ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｇｌｎ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１５）のいずれかのアミノ酸配列。
The calcium sensor protein of the present invention is a protein characterized by having the following amino acid sequences (a) to (k) in order from the N-terminus:
(A) the amino acid sequence represented by SEQ ID NO: 1;
(B) a sequence consisting of three amino acids Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each independently any amino acid) (linker A) (SEQ ID NO: 2);
(C) a myosin light chain kinase protein, or a partial amino acid sequence thereof comprising a calmodulin binding site;
(D) The sequence of (c) and the sequence of (e) described below are linked, Iso-Iso, Iso-Leu, Iso-Met, Iso-Pro, Iso-Val, Iso-Gly, Iso-Ala, Leu -Iso, Leu-Leu, Leu-Met, Leu-Pro, Leu-Val, Leu-Gly, Leu-Ala, Met-Iso, Met-Leu, Met-Met, Met-Pro, Met-Val, Met-Gly , Met-Ala, Pro-Iso, Pro-Leu, Pro-Met, Pro-Pro, Pro-Val, Pro-Gly, Pro-Ala, Val-Iso, Val-Leu, Val-Met, Val-Pro, Val -Val, Val-Gly, Val-Ala, Gly-Iso, Gly-Leu, Gly-Met, Gl Any selected from the group consisting of Pro, Gly-Val, Gly-Gly, Gly-Ala, Ala-Iso, Ala-Leu, Ala-Met, Ala-Pro, Ala-Val, Ala-Gly and Ala-Ala Any one amino acid sequence (linker B);
(E) the amino acid sequence from the Xth to the 236th amino acid sequence of the sequence shown in SEQ ID NO: 3, except for the 151st and / or 152th and / or 169th and / or 171st and / or 219th amino acids (Wherein X is an arbitrary position of 151 to 153);
(F) A sequence Gly-Gly-Xaa5-Gly-Gly-Xaa6 consisting of six amino acid sequences connecting the sequence of (e) and the sequence of (g) to be described later (where Xaa5 and Xaa6 are each independently arbitrary (SEQ ID NO: 4);
(G) an amino acid sequence from the first to the Y-th of the sequence represented by SEQ ID NO: 3,
Amino acid sequence in which the first and / or eighth and / or 52 and / or 54 and / or 76 and / or 136 amino acids are substituted with other amino acids (where Y is any of 144 to 150) Position);
(H) Amino acid sequence Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg or Thr (linker C) linking the sequence of (g) and the sequence of (i) described later );
(I) Amino acid sequence from 2nd to 148th of the sequence shown in SEQ ID NO: 9, except for the 63rd and / or 77th and / or 101st and / or 111th and / or 127th amino acids An amino acid sequence substituted with the amino acid of
(J) A sequence consisting of 15 amino acid sequences Gly-Gly-Gly-Xaa7-Gly-Gly-Xaa8-Gly-Gly-Gly-Gly, linking the sequence of (i) and the sequence of (k) described later -Gly-Gly-Xaa9-Xaa10 (where Xaa7, Xaa8, Xaa9 and Xaa10 are each independently any amino acid) (linker D) (SEQ ID NO: 10);
(K) Pro-Val-Lys-Gln-Thr-Leu-Asn-Phe-Asp-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 12 ), Gln-Cys-Thr-Asn-Tyr-Ala-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 13), Glu-Gly-Arg -Gly-Ser-Leu-Leu-Thr-Cys-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly (SEQ ID NO: 14) or Ala-Thr-Asn-Phe-Ser-Leu-Leu-Lys- Gln-Ala-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly Any of the amino acid sequences of the number 15).
ここで、配列（ａ）（配列番号１で示されるアミノ酸配列）は、５番目から１０番目がＨｉｓで構成されているが、このＨｉｓの数は、０〜６の間のいずれであってもよく、好ましくは６である。また、２番目のＡｒｇは存在していても、欠失していてもよい。
また、上記配列（ｅ）、（ｇ）及び（ｉ）は、少なくとも１の配列（（ｅ）、（ｇ）又は（ｉ））に上述のアミノ酸置換が含まれていればよく、例えば、配列（ｅ）の１５２番目と１６９番目のアミノ酸のみが置換され、配列（ｇ）が配列番号３で示される配列の１番目〜Ｙ番目までのアミノ酸配列であってアミノ酸の置換を含まず（ここで、Ｙは１４１〜１４８の任意の位置である）、配列（ｉ）が配列番号９で示される配列の２番目〜１４８番目までのアミノ酸配列であってアミノ酸の置換を含まないものであってもよい。
Here, the sequence (a) (amino acid sequence represented by SEQ ID NO: 1) is composed of His at the 5th to 10th positions, and the number of His may be any number between 0 and 6. Well, preferably 6. The second Arg may be present or may be deleted.
In addition, the sequences (e), (g) and (i) need only contain the above-mentioned amino acid substitution in at least one sequence ((e), (g) or (i)). Only the 152nd and 169th amino acids in (e) are substituted, and the sequence (g) is the first to Yth amino acid sequence of the sequence represented by SEQ ID NO: 3 and does not include amino acid substitution (here Y is an arbitrary position of 141 to 148), and the sequence (i) is an amino acid sequence from the 2nd to the 148th amino acid sequence of the sequence represented by SEQ ID NO: 9 and does not include amino acid substitution Good.
上記配列（ｋ）は、本発明のカルシウムセンサー蛋白質が核へ移行しないという性質を付与する働きをする。配列（ｋ）は、Ｌｅｕ−Ｘａａ１１−Ｘａａ１２−Ｘａａ１３−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｘａａ１４−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号２２）からなる配列をコンセンサス配列として含むものが望ましく、Ｘａａ１１はＫ又はＬ、Ｘａａ１２はＬ、Ｔ又はＱ、Ｘａａ１３はＡ又はＣ、Ｘａａ１４はＳ又はＥであることが好ましく、配列（ｋ）としては、例えば、配列番号１２〜１５に示される配列が好ましく、配列番号１２で示される配列が特に好ましい。 The sequence (k) functions to impart the property that the calcium sensor protein of the present invention does not move to the nucleus. The sequence (k) preferably includes a sequence consisting of Leu-Xaa11-Xaa12-Xaa13-Gly-Asp-Val-Glu-Xaa14-Asn-Pro-Gly (SEQ ID NO: 22) as a consensus sequence, and Xaa11 is K or L, Xaa12 is preferably L, T or Q, Xaa13 is preferably A or C, and Xaa14 is preferably S or E. As the sequence (k), for example, the sequences shown in SEQ ID NOs: 12 to 15 are preferable, The sequence represented by 12 is particularly preferred.
上記のカルシウムセンサー蛋白質は、主として、改変ｍＡｐｐｌｅ（ｅ及びｇが該当）、（２）機能性分子（ｃ及びｉが該当）、（３）リンカー（ｂ、ｄ、ｆ及びｈ）、（４）カルシウムセンサー蛋白質の核移行を抑制するペプチド（ｋが該当）から構成されている。 The calcium sensor protein is mainly composed of modified mapple (e and g), (2) functional molecule (c and i), (3) linker (b, d, f and h), (4) It consists of a peptide (k corresponds) that suppresses nuclear translocation of calcium sensor protein.
改変ｍＡｐｐｌｅとは、蛍光特性に影響を及ぼすホットスポットアミノ酸残基の近傍でｍＡｐｐｌｅのアミノ酸配列を切断して該蛋白質の構造を改変し、さらに特定部位のアミノ酸残基を置換したものである。 The modified mapple is a protein in which the amino acid sequence of the mapple is cleaved in the vicinity of the hot spot amino acid residue that affects the fluorescence properties to modify the protein structure, and the amino acid residue at a specific site is further substituted.
本発明のｍＡｐｐｌｅは、配列番号３で示されるアミノ酸配列からなる蛋白質である。また、本発明のｍＡｐｐｌｅのホモログとは、配列番号３で示されるアミノ酸と同一又は実質的に同一のアミノ酸配列を含む蛋白質のことである。ここで、「実質的に同一のアミノ酸配列を含む蛋白質」とは、配列番号３で示されるアミノ酸配列と約６０％以上、好ましくは約７０％以上、より好ましくは約８０％，８１％，８２％，８３％，８４％，８５％，８６％，８７％，８８％，８９％，９０％，９１％，９２％，９３％，９４％，９５％，９６％，９７％，９８％，最も好ましくは約９９％のアミノ酸同一性を有するアミノ酸配列を含み、かつ、蛍光を発する蛋白質のことであり、例えば、ＤｓＲｅｄ、ｍＣｈｅｒｒｙ、ｍＳｔｒａｗｂｅｒｒｙ、ｍＢａｎａｎａ、ｍＰｌｕｍ、ｔｄＴｏｍａｔｏ等のｍＡｐｐｌｅホモログがあげられる。 The mApple of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 3. In addition, the homolog of the mapple of the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid represented by SEQ ID NO: 3. Here, the “protein containing substantially the same amino acid sequence” is about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, or 82 with the amino acid sequence represented by SEQ ID NO: 3. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Most preferably, it is a protein that contains an amino acid sequence having about 99% amino acid identity and emits fluorescence, and examples thereof include mapple homologues such as DsRed, mCherry, mStrawberry, mBanana, mPlum, and tdTomato.
あるいは、配列番号３で示されるアミノ酸配列と実質的に同一のアミノ酸配列を含む蛋白質としては、配列番号３で表わされるアミノ酸配列中の１又は数個（好ましくは、１〜３０個程度、より好ましくは１〜１０個程度、さらに好ましくは１〜５個）のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、蛍光を発する蛋白質のことである。 Alternatively, as a protein comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3, one or several (preferably, about 1 to 30 amino acids, more preferably, in the amino acid sequence represented by SEQ ID NO: 3 Is a protein that consists of an amino acid sequence in which about 1 to 10 amino acids, more preferably 1 to 5 amino acids are deleted, substituted, or added, and emits fluorescence.
本発明において、ｍＡｐｐｌｅの蛍光特性に影響を及ぼす「ホットスポットアミノ酸残基」とは、改変ｍＡｐｐｌｅを作成する際に、その改変位置の指標となるｍＡｐｐｌｅ上のアミノ酸残基であり、該アミノ酸残基の近傍でｍＡｐｐｌｅの構造を改変することにより、所望の改変ｍＡｐｐｌｅを作成することを可能とするものである。本発明において推定されるｍＡｐｐｌｅのホットスポットアミノ酸残基は、配列番号３で示されるＧＦＰのアミノ酸配列中、第１５１番目のアミノ酸である。 In the present invention, the “hot spot amino acid residue” that affects the fluorescence properties of mapple is an amino acid residue on the mapple that serves as an index of the modified position when creating a modified mapple. By modifying the structure of the mapple in the vicinity of, it is possible to create a desired modified mapple. The hot spot amino acid residue of mapple estimated in the present invention is the 151st amino acid in the amino acid sequence of GFP represented by SEQ ID NO: 3.
ｍＡｐｐｌｅの構造を改変するとは、好ましくは、推定されるホットスポットアミノ酸残基の近傍（好ましくは、ホットスポットアミノ酸残基の前後５アミノ酸の範囲内の各位置）でｍＡｐｐｌｅを切断し、さらに切断部位から適切な数のアミノ酸（好ましくは１−１０個のアミノ酸）を除去し、必要に応じてｍＡｐｐｌｅ本来のＮ末端と本来のＣ末端とを適切なアミノ酸配列で連結することをいう。 Modifying the structure of mApple preferably cleaves mApple in the vicinity of the presumed hot spot amino acid residue (preferably each position within the range of 5 amino acids before and after the hot spot amino acid residue), An appropriate number of amino acids (preferably 1 to 10 amino acids) are removed from, and if necessary, the original N-terminus and the original C-terminus of mA Apple are connected with an appropriate amino acid sequence.
特定部位のアミノ酸残基を置換した改変ｍＡｐｐｌｅとは、配列番号３で示されるｍＡｐｐｌｅのアミノ酸配列上、特定部位のアミノ酸残基（好ましくは１番目及び／又は８番目及び／又は５２番目及び／又は５４番目及び／又は７６番目及び／又は１３６番目及び／又は１５１番目及び／又は１５２番目及び／又は１６９番目及び／又は１７１番目及び／又は２１９番目のアミノ酸残基）を、いずれかのアミノ酸に置換（例えば、１番目のアミノ酸をＩｓｏ、Ｌｅｕ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＬｅｕに置換し、８番目のアミノ酸をＧｌｎ、Ａｓｐ、Ｇｌｕ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＡｓｐに置換し、５２番目のアミノ酸をＡｒｇ、Ｈｉｓ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａ、特に好ましくは、Ｖａｌに置換し、５４番目のアミノ酸をＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｓｅｒ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＶａｌに置換し、７６番目のアミノ酸をＩｓｏ、Ｌｅｕ、Ｍｅｔ、ＰｒｏＧｌｙ若しくはＡｌａ、特に好ましくはＡｌａに置換し、１３６番目のアミノ酸をＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｉｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＰｒｏに置換し、１５１番目のアミノ酸をＩｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、Ｔｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＶａｌに置換し、１５２番目のアミノ酸をＴｙｒ、Ｐｈｅ、Ｔｒｐ、Ｔｈｒ、Ｓｅｒ、Ａｓｐ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＳｅｒに置換し、１６９番目のアミノ酸をＨｉｓ、Ｌｙｓ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＧｌｙに置換し、１７１番目のアミノ酸をＡｒｇ、Ｈｉｓ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＡｒｇに置換し、２１９番目のアミノ酸をＰｈｅ、Ｔｒｐ、Ｔｈｒ、Ｓｅｒ、Ｃｙｓ、Ｇｌｙ若しくはＡｌａ、特に好ましくはＣｙｓに置換）したｍＡｐｐｌｅを用いて、上記にあるように構造を改変したｍＡｐｐｌｅをいう。 The modified mAapple in which an amino acid residue at a specific site is substituted refers to an amino acid residue at a specific site (preferably at the 1st and / or 8th and / or 52nd and / or on the amino acid sequence of mApple shown in SEQ ID NO: 3. 54th and / or 76th and / or 136th and / or 151st and / or 152th and / or 169th and / or 171st and / or 219th amino acid residues) (For example, the first amino acid is replaced with Iso, Leu, Pro, Val, Gly or Ala, particularly preferably Leu, and the eighth amino acid is replaced with Gln, Asp, Glu, Gly or Ala, particularly preferably Asp. , Amino acid 52, Arg, His, Iso, Leu, Met, Pro, Val, Gly Or Ala, particularly preferably Val, and the 54th amino acid is replaced with Tyr, Phe, Trp, Ser, Iso, Leu, Met, Pro, Val, Gly or Ala, particularly preferably Val, and 76th Is replaced with Iso, Leu, Met, ProGly or Ala, particularly preferably Ala, and the 136th amino acid is Tyr, Phe, Trp, Thr, Iso, Leu, Met, Pro, Val, Gly or Ala, particularly preferred Is replaced with Pro, the 151st amino acid is replaced with Iso, Leu, Met, Pro, Val, Tyr, Phe, Trp, Thr, Gly or Ala, particularly preferably Val, and the 152nd amino acid is replaced with Tyr, Phe, Trp, Thr, Ser, Asp, Gly Is replaced with Ala, particularly preferably Ser, the 169th amino acid is replaced with His, Lys, Gly or Ala, particularly preferably Gly, and the 171st amino acid is replaced with Arg, His, Gly or Ala, particularly preferably Arg. A mapple having a structure modified as described above using a mapple substituted and substituted with the amino acid at position 219 by Phe, Trp, Thr, Ser, Cys, Gly or Ala, particularly preferably Cys).
改変ｍＡｐｐｌｅの特に好ましい例としては、配列番号３で示される配列の１５１番目〜２３６番目までのアミノ酸配列であって、１５１番目のアミノ酸をＶａｌに置換し、１５２番目のアミノ酸をＳｅｒに置換し、１６９番目のアミノ酸をＧｌｙに置換し、１７１番目のアミノ酸をＡｒｇに置換し、２１９番目のアミノ酸をＣｙｓに置換したアミノ酸配列をＮ末端側とし、配列番号３で示される配列の１番目〜１５０番目までのアミノ酸であって、１番目のアミノ酸をＬｅｕに置換し、８番目のアミノ酸をＡｓｐに置換し、５２番目のアミノ酸をＶａｌに置換し、５４番目のアミノ酸をＶａｌに置換し、７６番目のアミノ酸をＡｌａに置換し、１３６番目のアミノ酸をＰｒｏに置換したアミノ酸をＣ末端側として、適切なアミノ酸配列により連結したものが挙げられる。
なお、配列番号３において、５２番目と５４番目のアミノ酸置換については、本発明において初めて見出されたものである。他の位置のアミノ酸置換については、Zhao et al., Science 333:1888-1891 2011（非特許文献１）を参照した。
As a particularly preferred example of the modified mapple, the amino acid sequence from position 151 to position 236 of the sequence represented by SEQ ID NO: 3, wherein the 151st amino acid is substituted with Val, the 152nd amino acid is substituted with Ser, The amino acid sequence in which the 169th amino acid is replaced with Gly, the 171st amino acid is replaced with Arg, and the 219th amino acid is replaced with Cys is the N-terminal side. The first to 150th positions of the sequence shown in SEQ ID NO: 3 The first amino acid is replaced with Leu, the eighth amino acid is replaced with Asp, the 52nd amino acid is replaced with Val, the 54th amino acid is replaced with Val, the 76th amino acid is replaced with By replacing the amino acid with Ala and substituting the 136th amino acid with Pro for the C-terminal side, the appropriate amino acid sequence The concatenation, and the like.
In SEQ ID NO: 3, the 52nd and 54th amino acid substitutions were found for the first time in the present invention. For amino acid substitutions at other positions, refer to Zhao et al., Science 333: 1888-1891 2011 (Non-patent Document 1).
機能性分子とは、機能性分子自身が、該分子に作用する因子の結合等の作用により、立体構造に変化を起こし得る分子であって、改変ｍＡｐｐｌｅに連結することで、自身の立体構造の変化を該改変ｍＡｐｐｌｅに伝え得る分子のことである。この際、該機能性分子は、自身の立体構造の変化を前記改変ｍＡｐｐｌｅに伝えることにより、前記改変ｍＡｐｐｌｅの立体構造を変化させ、蛍光特性を変化させるように機能する。 A functional molecule is a molecule that itself can change its steric structure by the action of a factor that acts on the molecule, etc., and is linked to the modified mapple to Molecules that can transmit changes to the modified mApple. At this time, the functional molecule functions to change the three-dimensional structure of the modified mapple by changing its own three-dimensional structure to the modified mapple, thereby changing the fluorescence characteristics.
従って、機能性分子は、自身の立体構造の変化を改変ｍＡｐｐｌｅに伝達し改変ｍＡｐｐｌｅの構造に変化を及ぼし得る位置で、改変ｍＡｐｐｌｅに連結されている必要がある。従って機能性分子は、本来のｍＡｐｐｌｅ構造を改変した部分に近接して連結されていることが好ましい。具体的には、ｍＡｐｐｌｅを切断した位置にリンカー分子を介して連結されていることが好ましい。 Therefore, the functional molecule needs to be linked to the modified mapple at a position where it can transmit a change in its three-dimensional structure to the modified mapple and change the structure of the modified mapple. Therefore, it is preferable that the functional molecule is linked in the vicinity of a portion where the original mA Apple structure is modified. Specifically, it is preferable to be linked via a linker molecule to the position where the mapple has been cleaved.
このような機能性分子は、一分子であってもよいし、二分子以上であってもよい。二分子の場合、機能性分子に作用する因子は、まず一方の機能性分子の立体構造に変化を及ぼし、次いでその立体構造が改変ｍＡｐｐｌｅの立体構造に変化を起こさせる。本発明における機能性分子としては、カルモジュリン蛋白質とミオシン軽鎖キナーゼ蛋白質との組み合わせが挙げられる。 Such a functional molecule may be one molecule or two or more molecules. In the case of two molecules, factors acting on a functional molecule first change the three-dimensional structure of one functional molecule, and then the three-dimensional structure causes a change to the three-dimensional structure of the modified mAple. Examples of the functional molecule in the present invention include a combination of a calmodulin protein and a myosin light chain kinase protein.
また、改変ｍＡｐｐｌｅに連結する機能性分子は、その機能性分子が生体内で発現している通りの全構造を有する必要はなく、機能性分子に作用する因子が結合する部位のみを有する一部構造であってもよい。本発明における好ましい例としては、カルモジュリン蛋白質、及び配列番号７に示すカルモジュリン結合機能を有するミオシン軽鎖キナーゼの一部を機能性分子として挙げることができる。このような機能性分子を改変ｍＡｐｐｌｅに適式に連結することで、該融合蛋白質はカルシウムセンサーとして機能し得る。 In addition, the functional molecule linked to the modified mAple does not need to have the entire structure as the functional molecule is expressed in the living body, and has only a part to which a factor acting on the functional molecule binds. It may be a structure. Preferred examples of the present invention include a calmodulin protein and a part of a myosin light chain kinase having a calmodulin binding function shown in SEQ ID NO: 7 as functional molecules. By appropriately linking such a functional molecule to the modified mapple, the fusion protein can function as a calcium sensor.
ここで機能性分子として使用したラットカルモジュリン蛋白質の１番目のアミノ酸から１４８番目のアミノ酸配列を配列番号９に示す。この機能性分子においても、特定の部位のアミノ酸残基が置換されていることが望ましい。例えば、配列番号９で示される配列中、６３番目及び／又は７７番目及び／又は１０１番目及び／又は１１１番目及び／又は１２７番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列などを好ましいアミノ酸置換の例として挙げることができる。
なお、ここで示すアミノ酸の好ましい位置については、Zhao et al., Science 333:1888-1891 2011（非特許文献１）を参照した。
The amino acid sequence from the 1st amino acid to the 148th amino acid sequence of the rat calmodulin protein used as a functional molecule is shown in SEQ ID NO: 9. Also in this functional molecule, it is desirable that an amino acid residue at a specific site is substituted. For example, in the sequence represented by SEQ ID NO: 9, an amino acid sequence obtained by substituting the 63rd and / or 77th and / or 101st and / or 111th and / or 127th amino acid with another amino acid is preferable amino acid substitution As an example.
In addition, Zhao et al., Science 333: 1888-1891 2011 (nonpatent literature 1) was referred about the preferable position of the amino acid shown here.
リンカーとは、上記改変ｍＡｐｐｌｅの切断部位、該改変ｍＡｐｐｌｅと上記機能性分子間の連結部位、及び改変ｍＡｐｐｌｅと機能性分子を連結してなる蛋白質とそのＮ末端側にあるアミノ酸配列（本発明においては配列番号１で示されるアミノ酸配列）との連結部位に位置する数残基のアミノ酸からなるペプチドである。各リンカー分子を区別するべく、以下のような名称を付している。 The linker refers to the cleavage site of the modified mapple, the linking site between the modified mapple and the functional molecule, the protein obtained by linking the modified mapple and the functional molecule, and the amino acid sequence on the N-terminal side (in the present invention). Is a peptide consisting of several residues of amino acids located at the linking site to the amino acid sequence shown in SEQ ID NO: 1. In order to distinguish each linker molecule, the following names are given.
改変ｍＡｐｐｌｅと機能性分子からなる融合蛋白質全体のＮ末端に存在する配列番号１で示されるアミノ酸配列（上記配列（ａ））とミオシン軽鎖キナーゼ蛋白質又はカルモジュリン結合部位を含むその部分アミノ酸配列とを連結するリンカーをリンカーＡと称する。リンカーＡは、Ｍｅｔを含む任意のアミノ酸配列であるが、好ましくは１〜１０残基のアミノ酸ペプチドであり、より好ましくは、３つのアミノ酸からなる配列Ｍｅｔ−Ｘａａ１−Ｘａａ２（ここでＸａａ１及びＸａａ２はそれぞれ独立して任意のアミノ酸である；配列番号２）、さらに好ましくは、Ｍｅｔ−Ｇｌｙ−Ｔｈｒ（配列番号５）またはＭｅｔ−Ｖａｌ−Ａｓｐ（配列番号６）である。 An amino acid sequence represented by SEQ ID NO: 1 (the above-mentioned sequence (a)) present at the N-terminus of the entire fusion protein consisting of a modified mAapple and a functional molecule, and a partial amino acid sequence including a myosin light chain kinase protein or a calmodulin binding site The linker to be linked is called linker A. The linker A is an arbitrary amino acid sequence including Met, but is preferably an amino acid peptide having 1 to 10 residues, more preferably a sequence Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each composed of 3 amino acids). Each is independently an arbitrary amino acid; SEQ ID NO: 2), more preferably Met-Gly-Thr (SEQ ID NO: 5) or Met-Val-Asp (SEQ ID NO: 6).
改変ｍＡｐｐｌｅのＮ末端側と機能性分子を連結するリンカーはリンカーＢと称し、任意のアミノ酸配列であるが、好ましくは０〜１０残基のアミノ酸ペプチドであり、より好ましくは、２つのアミノ酸からなる配列Ｘａａ３−Ｘａａ４（ここでＸａａ３およびＸａａ４はいずれもＩｓｏ、Ｌｅｕ、Ｍｅｔ、Ｐｒｏ、Ｖａｌ、ＧｌｙおよびＡｌａからなる群から選択される何れか一のアミノ酸である）、さらに好ましくは、Ｐｒｏ−Ｖａｌである。 The linker that connects the functional molecule to the N-terminal side of the modified mAple is referred to as linker B, and is an arbitrary amino acid sequence, preferably an amino acid peptide of 0 to 10 residues, more preferably composed of two amino acids. The sequence Xaa3-Xaa4 (wherein Xaa3 and Xaa4 are all any one amino acid selected from the group consisting of Iso, Leu, Met, Pro, Val, Gly and Ala), more preferably Pro-Val is there.
改変ｍＡｐｐｌｅのＣ末端側と機能性分子を連結するリンカーはリンカーＣと称し、Ｔｈｒ−Ａｒｇ、Ｐｈｅ−Ａｒｇ、Ｔｒｐ−Ａｒｇ、Ｔｙｒ−Ａｒｇ、Ｇｌｙ−Ａｒｇ、Ａｌａ−Ａｒｇ又はＴｈｒであり、特に好ましくはＴｈｒ−Ａｒｇである。 The linker that connects the functional molecule to the C-terminal side of the modified mapple is referred to as linker C, and is preferably Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg, or Thr. Is Thr-Arg.
機能性分子のＣ末端側に連結するリンカーはリンカーＤと称し、１５個のアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ７−Ｇｌｙ−Ｇｌｙ−Ｘａａ８−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ９−Ｘａａ１０（ここでＸａａ７、Ｘａａ８、Ｘａａ９及びＸａａ１０はそれぞれ独立して任意のアミノ酸である）（配列番号１０）であり、特に好ましい例としては、Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｔｈｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｕ−Ｐｈｅ（配列番号１１）が挙げられる。 The linker linked to the C-terminal side of the functional molecule is referred to as linker D, and is a sequence consisting of 15 amino acids Gly-Gly-Gly-Xaa7-Gly-Gly-Xaa8-Gly-Gly-Gly-Gly-Gly-Gly -Xaa9-Xaa10 (where Xaa7, Xaa8, Xaa9 and Xaa10 are each independently any amino acid) (SEQ ID NO: 10), and a particularly preferred example is Gly-Gly-Gly-Thr-Gly-Gly. -Ser-Gly-Gly-Gly-Gly-Gly-Gly-Glu-Phe (sequence number 11) is mentioned.
なお、ｍＡｐｐｌｅの本来のＮ末端とＣ末端とを連結するアミノ酸ペプチドもリンカーであり、好ましくは、アミノ酸２〜１０残基からなるペプチドであり、Ｇｌｙを多く含むものが好ましく、さらに好ましくは、６つのアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｘａａ５−Ｇｌｙ−Ｇｌｙ−Ｘａａ６（ここでＸａａ５及びＸａａ６はそれぞれ独立して任意のアミノ酸である；配列番号４）であり、特に好ましい例としては、Ｇｌｙ−Ｇｌｙ−Ｔｈｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ（配列番号８）が挙げられる。 The amino acid peptide that connects the original N-terminal and C-terminal of mApple is also a linker, preferably a peptide consisting of 2 to 10 amino acids, preferably containing a large amount of Gly, more preferably 6 A sequence consisting of two amino acid sequences Gly-Gly-Xaa5-Gly-Gly-Xaa6 (where Xaa5 and Xaa6 are each independently any amino acid; SEQ ID NO: 4). Particularly preferred examples include Gly-Gly -Thr-Gly-Gly-Ser (SEQ ID NO: 8).
本発明でのカルシウムセンサー蛋白質とは、当該蛋白質に含まれる機能性分子の立体構造に影響を及ぼす因子であるカルシウムを作用させることで該機能性分子の立体構造に影響を与え、該立体構造の変化がカルシウムセンサー蛋白質に含まれる改変ｍＡｐｐｌｅの立体構造に影響を与えることで、該改変ｍＡｐｐｌｅの蛍光特性を可逆的に変化させる蛋白質をいう。この変化は、蛍光顕微鏡若しくはレーザー顕微鏡等で捉えることが出来る程度の変化をいい、好ましくは肉眼で捉えることが出来る程度の変化をいう。蛍光特性の変化が蛍光強度の変化である場合、蛍光の変化量ΔＦ／Ｆが、好ましくは、少なくとも０．１以上変化すること、より好ましくは１以上の範囲で変化することをいう。 The calcium sensor protein in the present invention affects the three-dimensional structure of the functional molecule by acting calcium, which is a factor affecting the three-dimensional structure of the functional molecule contained in the protein. This refers to a protein that reversibly changes the fluorescent properties of the modified mApple by affecting the three-dimensional structure of the modified mApple contained in the calcium sensor protein. This change refers to a change that can be detected with a fluorescence microscope or a laser microscope, and preferably a change that can be detected with the naked eye. When the change in fluorescence characteristics is a change in fluorescence intensity, it means that the fluorescence change amount ΔF / F preferably changes by at least 0.1 or more, more preferably changes by 1 or more.
本発明において蛍光特性とは、蛍光強度、蛍光波長、蛍光強度比、吸光度、吸光波長などの蛍光特性を指す。本発明では蛍光特性の一例として、蛍光強度を使用する。 In the present invention, the fluorescence characteristics refer to fluorescence characteristics such as fluorescence intensity, fluorescence wavelength, fluorescence intensity ratio, absorbance, and absorption wavelength. In the present invention, fluorescence intensity is used as an example of fluorescence characteristics.
本発明において、蛍光特性が蛍光強度である場合、カルシウムセンサー蛋白質は、蛍光を発する状態と蛍光を発しない状態の臨界状態にある。この臨界状態においてカルシウムセンサー蛋白質は、蛍光を発しない状態にあってもよいし、蛍光強度の低い状態にあってもよい。あるいは蛍光強度の高い状態にあってもよい。ここで、蛍光を発しないとは、光学機器を使用して蛍光を確認できないことをいう。蛍光強度が低いとは、カルシウムの存在により、上記の変化量（ΔＦ／Ｆが少なくとも０．１以上変化する量）を示して、蛍光強度が高い状態に変化し得る程度に低いことをいう。同様に、蛍光強度が高いとは、カルシウムの存在により、上記の変化量（ΔＦ／Ｆが少なくとも０．１以上変化する量）を示して、蛍光強度が低い状態に変化し得る程度に高いことをいう。 In the present invention, when the fluorescence characteristic is fluorescence intensity, the calcium sensor protein is in a critical state of a state of emitting fluorescence and a state of not emitting fluorescence. In this critical state, the calcium sensor protein may be in a state where it does not emit fluorescence, or may be in a state where fluorescence intensity is low. Or you may exist in a state with high fluorescence intensity. Here, not emitting fluorescence means that fluorescence cannot be confirmed using an optical device. “Low fluorescence intensity” means the above change amount (amount that ΔF / F changes by at least 0.1 or more) due to the presence of calcium and is low enough to change the fluorescence intensity to a high state. Similarly, high fluorescence intensity indicates the amount of change described above (the amount by which ΔF / F changes by at least 0.1) due to the presence of calcium, and is high enough to change the fluorescence intensity to a low state. Say.
本発明におけるカルシウムセンサー蛋白質は、従来のカルシウムセンサー蛋白質に比べ、上記のように配列（ａ）、改変ｍＡｐｐｌｅ及びラットカルモジュリンの特定部位のアミノ酸残基を置換し、上記のような特定のリンカーを用いることにより、カルシウム作用時に、より大きな蛍光特性の変化を引き起こすことを特徴とする。ここでより大きな蛍光特性変化とは、蛍光特性の変化が蛍光強度の変化である場合、蛍光の変化量ΔＦ／Ｆが、従来のカルシウムセンサー蛋白質よりも大きく、好ましくは、２倍以上増強されることをいう。 Compared with the conventional calcium sensor protein, the calcium sensor protein in the present invention substitutes the amino acid residues at specific sites of the sequence (a), modified mApple and rat calmodulin as described above, and uses the specific linker as described above. Thus, it is characterized in that it causes a greater change in fluorescence characteristics during calcium action. Here, the larger change in fluorescence characteristic means that when the change in fluorescence characteristic is a change in fluorescence intensity, the amount of change ΔF / F in fluorescence is larger than that of the conventional calcium sensor protein, and is preferably enhanced two times or more. That means.
融合蛋白質の作成は、公知の遺伝子工学的手法を用いて行うことができる。例えば、融合したい各蛋白質部分をコードする遺伝子（即ち、特定アミノ酸部位の弛緩を伴う改変ｍＡｐｐｌｅ及び機能性分子をコードする遺伝子）の断片をそれぞれＰＣＲにより作成し、これら断片を繋ぎ合わせることにより融合遺伝子を作成し、次いで、該融合遺伝子を含むプラスミドを所望の細胞に導入して発現させることにより、融合蛋白質は作られる。 The fusion protein can be prepared using a known genetic engineering technique. For example, a fragment of a gene encoding each protein part to be fused (that is, a gene encoding a modified mapple with relaxation of a specific amino acid site and a functional molecule) is prepared by PCR, and these fragments are joined together to form a fusion gene. Then, a fusion protein is produced by introducing a plasmid containing the fusion gene into a desired cell and expressing it.
また、本発明においてカルシウムセンサー遺伝子は、本発明のカルシウムセンサー蛋白質をコードする遺伝子のことをいう。該カルシウムセンサー遺伝子は、上記にあるように、作成したいカルシウムセンサー蛋白質を構成する各構成部分をコードする遺伝子断片をそれぞれＰＣＲにより作成し、次いで、これら各遺伝子断片を連結させることにより、融合遺伝子の形で作成され得る。 In the present invention, the calcium sensor gene refers to a gene encoding the calcium sensor protein of the present invention. As described above, the calcium sensor gene is prepared by PCR by generating gene fragments encoding each component constituting the calcium sensor protein to be prepared, and then linking these gene fragments to form a fusion gene. Can be created in shape.
本発明において作成されたカルシウムセンサー蛋白質は、細胞内及び細胞外でカルシウム濃度をより安定に、かつ高感度に測定することが出来る。 The calcium sensor protein prepared in the present invention can measure calcium concentration more stably and with high sensitivity inside and outside the cell.
例えば、本発明のカルシウムセンサー遺伝子を大腸菌などに導入して予め産生されたカルシウムセンサー蛋白質と検体とを混合することによりカルシウム濃度を測定することが可能である。また、大腸菌などを使用して産生させた蛋白質を、カルシウム濃度を測定したい細胞に直接注入することにより、細胞内のカルシウム濃度を測定することも可能である。あるいは、本発明のカルシウムセンサー遺伝子を、カルシウム濃度を測定したい細胞に導入して細胞に蛋白質を産生させることにより、細胞内のカルシウム濃度を測定することも可能である。 For example, it is possible to measure the calcium concentration by introducing the calcium sensor gene of the present invention into Escherichia coli or the like and mixing a calcium sensor protein produced in advance with a specimen. It is also possible to measure the intracellular calcium concentration by directly injecting a protein produced using Escherichia coli or the like into the cell whose calcium concentration is to be measured. Alternatively, the intracellular calcium concentration can be measured by introducing the calcium sensor gene of the present invention into a cell whose calcium concentration is to be measured and causing the cell to produce a protein.
カルシウム濃度の測定は、ある特定の波長の光（例えば、５６８ｎｍの励起光）をカルシウムセンサー蛋白質に当てることにより、該蛋白質の発する蛍光特性を光学機器（例えば、レーザー顕微鏡）で検出することにより行う。なお、濃度測定に使用するカルシウムセンサー蛋白質が、どのくらいのカルシウム濃度でどのような蛍光特性を示すのか、予め調べておくことが必要である。具体的には、例えば、大腸菌により産生したカルシウムセンサー蛋白質により、蛍光分光光度計を用いて種々のカルシウム濃度に対する蛍光強度変化を測定しておくことが必要である（例えば、図４を参照のこと）。本測定により、Ｋｄ１７４ｎＭ、Ｈｉｌｌ係数１．５８、最大蛍光変化量２８．７という結果を得た。 The calcium concentration is measured by applying light of a specific wavelength (for example, excitation light of 568 nm) to the calcium sensor protein, and detecting the fluorescence characteristics emitted by the protein with an optical instrument (for example, a laser microscope). . In addition, it is necessary to investigate in advance what fluorescence characteristics the calcium sensor protein used for concentration measurement shows at what calcium concentration. Specifically, it is necessary to measure changes in fluorescence intensity with respect to various calcium concentrations using, for example, a calcium sensor protein produced by Escherichia coli using a fluorescence spectrophotometer (see, for example, FIG. 4). ). By this measurement, the results of Kd 174 nM, Hill coefficient 1.58, and maximum fluorescence change 28.7 were obtained.
以下に本発明の実施例を示すが、これらの実施例はあくまでも例示にすぎず、本発明の範囲を限定するものではない。 Examples of the present invention will be shown below, but these examples are merely illustrative and do not limit the scope of the present invention.
本発明のカルシウムセンサー蛋白質であるＲ−ＣａＭＰ１．０７の特性
本発明のカルシウムセンサー蛋白質であるＲ−ＣａＭＰ１．０７、その前駆体であるＲ−ＣａＭＰ１．０１および従来のカルシウムセンサー蛋白質であるＲ−ＧＥＣＯ１について、これらの蛋白質をコードする遺伝子をヒト子宮癌由来株化細胞であるＨｅＬａ細胞に導入して、カルシウムセンサーとしての性能を評価した。まず細胞内局在部位について検討した結果、Ｒ−ＧＥＣＯ１およびＲ−ＣａＭＰ１．０１はいずれも細胞質と核内への局在を示したのに対し、Ｒ−ＣａＭＰ１．０７は細胞質には局在するが核内には局在しないことがわかった（図１）。
Characteristics of R-CaMP1.07, a calcium sensor protein of the present invention R-CaMP1.07, a calcium sensor protein of the present invention, R-CaMP1.01 as a precursor thereof, and R-GECO1 as a conventional calcium sensor protein The genes encoding these proteins were introduced into HeLa cells, which are human uterine cancer-derived cell lines, and their performance as calcium sensors was evaluated. First, as a result of examining the intracellular localization site, R-GECO1 and R-CaMP1.01 both showed localization in the cytoplasm and nucleus, whereas R-CaMP1.07 was localized in the cytoplasm. Was not localized in the nucleus (FIG. 1).
次いでＲ−ＧＥＣＯ１、Ｒ−ＣａＭＰ１．０１およびＲ−ＣａＭＰ１．０７を発現させたＨｅＬａ細胞に、該細胞の細胞内カルシウムイオン濃度を増大させることが既知である、ＡＴＰ（０．１ｍＭ）を作用させた。その時の反応例を図２に示す。従来のカルシウムセンサー蛋白質Ｒ−ＧＥＣＯ１よりも本発明のカルシウムセンサー蛋白質であるＲ−ＣａＭＰ１．０７およびその前駆体であるＲ−ＣａＭＰ１．０１の方が、大きな蛍光強度の上昇を示すことがわかった。 Next, ATP (0.1 mM), which is known to increase intracellular calcium ion concentration, is allowed to act on HeLa cells expressing R-GECO1, R-CaMP1.01 and R-CaMP1.07. It was. An example of the reaction at that time is shown in FIG. It was found that R-CaMP1.07 which is the calcium sensor protein of the present invention and R-CaMP1.01 which is a precursor thereof show a larger increase in fluorescence intensity than the conventional calcium sensor protein R-GECO1.
本発明のカルシウムセンサー蛋白質Ｒ−ＣａＭＰ１．０７およびその前駆体であるＲ−ＣａＭＰ１．０１を発現させたＨｅＬａ細胞でのＡＴＰに対する蛍光強度の変化量は、従来のカルシウムセンサー蛋白質Ｒ−ＧＥＣＯ１のそれに比して両者とも約１．７倍大きいことを確認した。（図３）。
さらに、本発明のカルシウムセンサー蛋白質Ｒ−ＣａＭＰ１．０７およびその前駆体Ｒ−ＣａＭＰ１．０１の精製蛋白質溶液、およびＲ−ＧＥＣＯ１を含む細胞破砕液を用いて試験管レベルでカルシウム濃度と蛍光量との関係を検討した結果、Ｒ−ＣａＭＰ１．０７およびＲ−ＣａＭＰ１．０１は、Ｒ−ＧＥＣＯ１と比較すると、カルシウムイオンの有無での蛍光強度変化量が各々約２倍および約１．４倍大きい（カルシウムイオンの有無での蛍光強度の比率を求めると、Ｒ−ＣａＭＰ１．０７は２８．７倍でありＲ−ＣａＭＰ１．０１は２０．３倍であるのに対し、Ｒ−ＧＥＣＯ１は１４．２倍である）ことを確認した（図４）。
このように本発明のカルシウムセンサー蛋白質Ｒ−ＣａＭＰ１．０７およびその前駆体Ｒ−ＣａＭＰ１．０１は、従来のカルシウムセンサー蛋白質Ｒ−ＧＥＣＯ１に比して、より高い蛍光反応性を示すことが証明されている。またＲ−ＣａＭＰ１．０７は、Ｒ−ＣａＭＰ１．０１およびＲ−ＧＥＣＯ１と異なり、細胞に発現させた場合に細胞質には局在するが核内には局在しないという、細胞質カルシウム濃度測定に適した局在パターンを示すことが証明されている。
The amount of change in fluorescence intensity with respect to ATP in HeLa cells in which the calcium sensor protein R-CaMP1.07 and its precursor R-CaMP1.01 of the present invention were expressed was compared with that of the conventional calcium sensor protein R-GECO1. It was confirmed that both were about 1.7 times larger. (Figure 3).
Furthermore, the calcium concentration and fluorescence amount at the test tube level using the purified protein solution of the calcium sensor protein R-CaMP1.07 and its precursor R-CaMP1.01 of the present invention and the cell lysate containing R-GECO1. As a result of investigating the relationship, R-CaMP1.07 and R-CaMP1.01 have about two times and about 1.4 times larger fluorescence intensity changes in the presence or absence of calcium ions, respectively, than R-GECO1 (calcium When the ratio of the fluorescence intensity with and without ions is determined, R-CaMP1.07 is 28.7 times and R-CaMP1.01 is 20.3 times, whereas R-GECO1 is 14.2 times. (Fig. 4).
Thus, it has been proved that the calcium sensor protein R-CaMP1.07 and its precursor R-CaMP1.01 of the present invention exhibit higher fluorescence reactivity than the conventional calcium sensor protein R-GECO1. Yes. Moreover, unlike R-CaMP1.01 and R-GECO1, R-CaMP1.07 is suitable for the measurement of cytoplasmic calcium concentration, which is localized in the cytoplasm when expressed in cells but not in the nucleus. It has been proven to show a localized pattern.
カルシウムセンサー蛋白質Ｒ−ＣａＭＰ１．０７の製法及び測定法
（Ａ）Ｒ−ＣａＭＰ１．０７の製法
（Ａ−１）細菌発現用および哺乳動物発現用のプラスミド構築
Ｒ−ＣａＭＰ１．０７の細菌発現用プラスミドであるｐＲＳＥＴＢ−Ｒ−ＣａＭＰ１．０７及び哺乳動物発現用プラスミドであるｐＮ１−Ｒ−ＣａＭＰ１．０７は、非特許文献１（Science 333(6051):1888-1891,2011）に記載のＲ−ＧＥＣＯ１（配列番号２４）をコードするｃＤＮＡ（配列番号２５）を用いて後述のように構築した。
すなわち、まずＲ−ＧＥＣＯ１をコードするｃＤＮＡを化学的な遺伝子合成により構築し、そのｃＤＮＡを用いてＲ−ＧＥＣＯ１の細菌発現用プラスミドｐＲＳＥＴＢ―Ｒ−ＧＥＣＯ１および哺乳動物発現用プラスミドｐＮ１―Ｒ−ＧＥＣＯ１を構築した。次にＲ−ＧＥＣＯ１の蛋白質精製を可能にするため、Ｒ−ＧＥＣＯ１のＮ末端にＲＳＥＴタグ（蛋白質精製用のＨｉｓ6タグを含む）（配列番号２３）を付加したクローンであるｄＲＧｅｃの細菌発現用プラスミドｐＲＳＥＴＢ―ｄＲＧｅｃを構築した。さらに蛍光変化量の大きいｄＲＧｅｃの変異体の開発を目指して、ｐＲＳＥＴＢ―ｄＲＧｅｃをテンプレートとしたランダムＰＣＲによりｄＲＧｅｃの改変ｍＡｐｐｌｅ部分にさまざまな変異を導入した変異体を作製した。このＰＣＲ産物で大腸菌ＫＲＸを形質転換して明るく赤色蛍光を発し、かつカルシウムの有無での明るさの差が大きいｄＲＧｅｃの変異体クローンをスクリーニングした結果、ｄＲＧｅｃ（Ｋ５２Ｖ／Ｔ５４Ｖ）を発見した。このクローンをＲ−ＣａＭＰ１．０１（配列番号１６（アミノ酸配列）、配列番号１７（核酸配列））と命名した。次にＲ−ＣａＭＰ１．０１の細菌発現用プラスミドであるｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０１を用い、Ｒ−ＣａＭＰ１．０１の哺乳動物発現用プラスミドであるｐＮ１―Ｒ−ＣａＭＰ１．０１を構築した。
Method for producing and measuring calcium sensor protein R-CaMP1.07 (A) Method for producing R-CaMP1.07 (A-1) Plasmid construction for bacterial expression and mammalian expression Plasmid for bacterial expression of R-CaMP1.07 PRSET B -R-CaMP1.07 and pN1-R-CaMP1.07 which is a plasmid for mammalian expression are R-GECO1 (Non-patent Document 1 (Science 333 (6051): 1888-1891, 2011)). It was constructed as described below using cDNA (SEQ ID NO: 25) encoding SEQ ID NO: 24).
That is, first, cDNA encoding R-GECO1 is constructed by chemical gene synthesis, and R-GECO1 bacterial expression plasmid pRSET B- R-GECO1 and mammalian expression plasmid pN1-R-GECO1 are used by using the cDNA. Built. Next, in order to enable protein purification of R-GECO1, a plasmid for bacterial expression of dRGec, which is a clone with an RSET tag (including His6 tag for protein purification) (SEQ ID NO: 23) added to the N-terminus of R-GECO1 pRSET B -dRGec was constructed. Furthermore, with the aim of developing mutants of dRGec with a large amount of fluorescence change, mutants were prepared by introducing various mutations into the modified mApple part of dRGec by random PCR using pRSET B- dRGec as a template. Escherichia coli KRX was transformed with this PCR product and bright red fluorescence was emitted, and mutant mutants of dRGec having a large difference in brightness with and without calcium were screened. As a result, dRGec (K52V / T54V) was discovered. This clone was named R-CaMP1.01 (SEQ ID NO: 16 (amino acid sequence), SEQ ID NO: 17 (nucleic acid sequence)). Next, pN1-R-CaMP1.01, which is a plasmid for mammalian expression of R-CaMP1.01, was constructed using pRSET B- R-CaMP1.01, which is a plasmid for bacterial expression of R-CaMP1.01.
次に、カルシウムセンサー蛋白質の構造は一般的に非常に繊細であり、その性能がＮ末端やＣ末端へのペプチド融合により修飾されることが知られていることから、Ｒ−ＣａＭＰ１．０１の改良の一環として、該センサー蛋白質へのＮ末端やＣ末端へのペプチド融合を検討した。ここではＲ−ＣａＭＰ１．０１のC末端への１５アミノ酸のリンカー配列（配列番号１１）およびＦ２Ａ配列（配列番号１２）で構成されるペプチドを融合させた実施例を述べる。１５アミノ酸のリンカー配列（配列番号１１）およびＦ２Ａ配列（配列番号１２）をＣ末端に付加させたＲ−ＣａＭＰ１．０１改変体をＲ−ＣａＭＰ１．０７（配列番号１８（アミノ酸配列）、配列番号１９（核酸配列））と命名し、その細菌発現用プラスミドｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０７および哺乳動物発現用プラスミドｐＮ１―Ｒ−ＣａＭＰ１．０７を構築した。 Next, since the structure of the calcium sensor protein is generally very delicate and its performance is known to be modified by peptide fusion to the N-terminus or C-terminus, improvement of R-CaMP1.01 As part of this, peptide fusion to the N-terminal and C-terminal to the sensor protein was examined. Here, an example in which a peptide composed of a 15 amino acid linker sequence (SEQ ID NO: 11) and an F2A sequence (SEQ ID NO: 12) to the C-terminus of R-CaMP1.01 is described. An R-CaMP1.01 variant in which a 15-amino acid linker sequence (SEQ ID NO: 11) and an F2A sequence (SEQ ID NO: 12) were added to the C-terminus was converted to R-CaMP1.07 (SEQ ID NO: 18 (amino acid sequence), SEQ ID NO: 19 (Nucleic acid sequence)) and the bacterial expression plasmid pRSET B- R-CaMP1.07 and the mammalian expression plasmid pN1-R-CaMP1.07 were constructed.
より具体的には、１番目にまずＲ−ＧＥＣＯ１のｃＤＮＡを化学合成（Ｇｅｎｓｃｒｉｐｔ）し、これをＮｄｅＩ−ＮｏｔＩで消化した１．２６ｋｂの断片をｐＲＳＥＴＢ―Ｇ−ＣａＭＰ６（特許文献３）をＮｄｅＩ−ＮｏｔＩで消化した２．７７ｋｂの断片とライゲーションさせてＲ−ＧＥＣＯ１の細菌発現用プラスミドであるｐＲＳＥＴＢ―Ｒ−ＧＥＣＯ１を構築した。またｐＲＳＥＴＢ―Ｒ−ＧＥＣＯ１をＳａｌＩ−ＮｏｔＩで消化した１．２５ｋｂの断片をｐＮ１―Ｇ−ＣａＭＰ（特許文献１）をＳａｌＩ−ＮｏｔＩで消化した３．９６ｋｂの断片とライゲーションさせてＲ−ＧＥＣＯ１の哺乳動物発現用プラスミドであるｐＮ１―Ｒ−ＧＥＣＯ１を構築した。次にｐＮ１―Ｒ−ＧＥＣＯ１をＳａｌＩ−ＮｏｔＩで消化した１．２５ｋｂの断片をｐＲＳＥＴＢ―Ｇ−ＣａＭＰ６（特開２０１２−８５５４２）をＳａｌＩ−ＮｏｔＩで消化した２．８８ｋｂの断片とライゲーションさせてＲ−ＧＥＣＯ１のＮ末端にＲＳＥＴタグ（配列番号２３）を付加したクローンであるｄＲＧｅｃの細菌発現用プラスミドであるｐＲＳＥＴＢ―ｄＲＧｅｃを構築した。 More specifically, R-GECO1 cDNA was first chemically synthesized (Genscript), and a 1.26 kb fragment obtained by digesting this with NdeI-NotI was converted into pRSET B -G-CaMP6 (Patent Document 3). -PRSET B -R-GECO1, which is a plasmid for bacterial expression of R-GECO1, was constructed by ligation with a 2.77 kb fragment digested with NotI. In addition, a 1.25 kb fragment obtained by digesting pRSET B- R-GECO1 with SalI-NotI was ligated with a 3.96 kb fragment obtained by digesting pN1-G-CaMP (Patent Document 1) with SalI-NotI. PN1-R-GECO1, a plasmid for mammalian expression, was constructed. Next, the 1.25 kb fragment obtained by digesting pN1-R-GECO1 with SalI-NotI was ligated with the 2.88 kb fragment obtained by digesting pRSET B- G-CaMP6 (Japanese Patent Laid-Open No. 2012-85542) with SalI-NotI. -Construction of pRSET B -dRGec, which is a plasmid for bacterial expression of dRGec, a clone with a RSET tag (SEQ ID NO: 23) added to the N-terminus of GECO1.
２番目に、ｐＲＳＥＴＢ―ｄＲＧｅｃをテンプレートとして以下の合成プライマー（Ｏｐｅｒｏｎ）
５’−AGCTATAGGTCGGCTGAGCTCA−３’（Ｍ１３（Ｓａｃａ））（配列番号２０）
５’−CTTCAGTCAGTTGGTCACGCGT−３’（ｒＣａＭ（Ｍｌｕ））（配列番号２１）
を用いてＰＣＲによるランダム変異導入を後述の方法で行った。このＰＣＲ産物をＳａｃＩとＭｌｕＩで消化した０．７４ｋｂの断片をＳａｃＩとＭｌｕＩで消化したｐＲＳＥＴＢ―ｄＲＧｅｃの３．３９ｋｂの断片とライゲーションさせてｐＲＳＥＴＢ―ｄＲＧｅｃ（ｒａｎｄｏｍ）を構築した。ｐＲＳＥＴＢ―ｄＲＧｅｃ（ｒａｎｄｏｍ）で大腸菌ＫＲＸを形質転換して明るく赤色蛍光を発し、かつカルシウムの有無での明るさの差が大きいｄＲＧｅｃの変異体クローンをスクリーニングした結果、ｄＲＧｅｃの改良体であるｄＲＧｅｃ（Ｋ５２Ｖ／Ｔ５４Ｖ）を発見した。このクローンをＲ−ＣａＭＰ１．０１（配列番号１６（アミノ酸配列）、配列番号１７（核酸配列））と命名した。Ｒ−ＣａＭＰ１．０１の細菌発現用プラスミドであるｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０１をＳａｌＩとＮｏｔＩで消化した１．２５ｋｂの断片をｐＮ１―Ｇ−ＣａＭＰ６（特許文献３）をＳａｌＩとＮｏｔＩで消化した４．０６ｋｂの断片とライゲーションさせてＲ−ＣａＭＰ１．０１の哺乳動物発現用プラスミドであるｐＮ１−Ｒ−ＣａＭＰ１．０１を構築した。
Second, the following synthetic primer (Operon) using pRSET B- dRGec as a template
5′-AGCTATAGGTCGGCTGAGCTCA-3 ′ (M13 (Saca)) (SEQ ID NO: 20)
5′-CTTCAGTCAGTTGGTCACGCGT-3 ′ (rCaM (Mlu)) (SEQ ID NO: 21)
Random mutagenesis by PCR was carried out by the method described later. The PCR product was digested with SacI and MluI, and the 0.74 kb fragment was ligated with the 3.39 kb fragment of pRSET B- dRGec digested with SacI and MluI to construct pRSET B- dRGec (random). Escherichia coli KRX was transformed with pRSET B -dRGec (random), and a mutant clone of dRGec that emitted bright red fluorescence and had a large difference in brightness with or without calcium was screened. (K52V / T54V) was discovered. This clone was named R-CaMP1.01 (SEQ ID NO: 16 (amino acid sequence), SEQ ID NO: 17 (nucleic acid sequence)). PRSET B -R-CaMP1.01, which is a plasmid for bacterial expression of R-CaMP1.01, was digested with SalI and NotI. A 1.25 kb fragment was digested with pN1-G-CaMP6 (Patent Document 3) with SalI and NotI. PN1-R-CaMP1.01, which is a plasmid for mammalian expression of R-CaMP1.01, was constructed by ligation with the 4.06 kb fragment.
３番目に、カルモジュリンのＣ末端部分（１２９〜１４８番目のアミノ酸を含む）、１５アミノ酸のリンカー配列（配列番号１１）およびＦ２Ａ配列（配列番号１２）を含むｃＤＮＡを化学合成（Ｇｅｎｓｃｒｉｐｔ）し、これをＣｌａＩ−ＮｏｔＩで消化した０．１７ｋｂの断片をｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０１をＣｌａＩ−ＮｏｔＩで消化した４．０７ｋｂの断片とライゲーションさせて１５アミノ酸のリンカー配列およびＦ２Ａ配列をＣ末端に付加させたＲ−ＣａＭＰ１．０１改変体であるＲ−ＣａＭＰ１．０７の細菌発現用プラスミドであるｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０７を構築した。ｐＲＳＥＴＢ―Ｒ−ＣａＭＰ１．０７をＳａｌＩとＮｏｔＩで消化した１．３６ｋｂの断片をｐＮ１―Ｇ−ＣａＭＰ６（特許文献３）をＳａｌＩとＮｏｔＩで消化した４．０６ｋｂの断片とライゲーションさせてＲ−ＣａＭＰ１．０７の哺乳動物発現用プラスミドであるｐＮ１−Ｒ−ＣａＭＰ１．０７を構築した。 Third, a cDNA containing the C-terminal part of calmodulin (including amino acids 129 to 148), 15 amino acid linker sequence (SEQ ID NO: 11) and F2A sequence (SEQ ID NO: 12) was chemically synthesized (Genscript). The 0.17 kb fragment digested with ClaI-NotI was ligated with the 4.07 kb fragment digested with pRSET B- R-CaMP1.01 with ClaI-NotI to add a 15 amino acid linker sequence and F2A sequence to the C-terminus PRSET B -R-CaMP1.07, which is a plasmid for bacterial expression of R-CaMP1.07, which is a modified version of R-CaMP1.01, was constructed. A 1.36 kb fragment obtained by digesting pRSET B- R-CaMP1.07 with SalI and NotI was ligated with a 4.06 kb fragment obtained by digesting pN1-G-CaMP6 (Patent Document 3) with SalI and NotI to obtain R-CaMP1. PN1-R-CaMP1.07, a mammalian expression plasmid of 0.07, was constructed.
ＰＣＲによるランダム変異導入は以下のように行った。すなわち詳細には、まずテンプレートのプラスミドを１μｇ、１０ｘバッファーを５μｌ、２．５ｍＭのｄＮＴＰを１μｌ、１０μＭのフォワードプライマー（配列番号２０）を１μｌ、１０μＭのリバースプライマー（配列番号２１）を１μｌ、５ｍMのＭｎＣｌ2を１．５μｌ、Ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｔａｋａｒａ）を１μｌ、水を加えて全量を４７μｌとした混合液を４つ用意した。４つの混合液にはさらに各々１）１０ｍＭのｄＴＴＰを１μｌ、１０ｍＭのｄＧＴＰを１μｌ、１０ｍＭのｄＣＴＰを１μｌ、または２）１０ｍＭのｄＡＴＰを１μｌ、１０ｍＭのｄＧＴＰを１μｌ、１０ｍＭのｄＣＴＰを１μｌ、または３）１０ｍＭのｄＡＴＰを１μｌ、１０ｍＭのｄＴＴＰを１μｌ、１０ｍＭのｄＣＴＰを１μｌ、または４）１０ｍＭのｄＡＴＰを１μｌ、１０ｍＭのｄＴＴＰを１μｌ、１０ｍＭのｄＧＴＰを１μｌ、を加えて各々全量を５０μｌとし、下記の条件に付した。 Random mutagenesis by PCR was performed as follows. Specifically, first, 1 μg of template plasmid, 5 μl of 10 × buffer, 1 μl of 2.5 mM dNTP, 1 μl of 10 μM forward primer (SEQ ID NO: 20), 1 μl of 10 μM reverse primer (SEQ ID NO: 21), 5 mM Four MnCl 2 , 1.5 μl Taq DNA polymerase (Takara) 1 μl, and water were added to prepare a total of 47 μl. Each of the four mixtures further includes 1) 1 μl of 10 mM dTTP, 1 μl of 10 mM dGTP, 1 μl of 10 mM dCTP, or 2) 1 μl of 10 mM dATP, 1 μl of 10 mM dGTP, or 1 μl of 10 mM dCTP, or 3) 1 μl of 10 mM dATP, 1 μl of 10 mM dTTP, 1 μl of 10 mM dCTP, or 4) 1 μl of 10 mM dATP, 1 μl of 10 mM dTTP, 1 μl of 10 mM dGTP, The following conditions were applied.
ステップ１
摂氏９４度 ２分
ステップ２
摂氏９４度 ３０秒
摂氏５５度 ３０秒
摂氏６８度 ３０秒
上記を３８サイクル
ステップ３
摂氏６８度 １分
上記の４つの混合液から各々２．５μｌずつを取って混合した１０μｌを下記の方法でアガロース電気泳動に付し、ＰＣＲ産物の確認を行った。
Step 1
94 degrees Celsius 2 minutes step 2
94 degrees Celsius 30 seconds 55 degrees Celsius 30 seconds 68 degrees Celsius 30 seconds
68 degrees Celsius, 1 minute Each 10 μl of 2.5 μl of each of the above four mixtures was mixed and subjected to agarose electrophoresis by the following method to confirm the PCR product.
制限酵素によるＤＮＡの切断はＮＥＢ社、Ｔｏｙｏｂｏ社、またはＴａｋａｒａ社の制限酵素、およびその添付バッファーと添付Ｂｏｖｉｎｅ Ｓｅｒｕｍ Ａｌｂｕｍｉｎ（１００ｘＢＳＡ）を用いて行った。反応は、１〜２μｇのＤＮＡに添付バッファー（３μｌ）、添付１００ｘＢＳＡ（０．３μｌ）および各制限酵素（１０ユニット）を加えて全量を３０μｌとした中で、摂氏３７度で１〜３時間行った。 Cleavage of DNA with restriction enzymes was performed using NEB, Toyobo, or Takara restriction enzymes, and their attached buffers and attached Bovine Serum Albumin (100 × BSA). The reaction was performed at 37 ° C. for 1 to 3 hours by adding 1 to 2 μg of DNA to the attached buffer (3 μl), attached 100 × BSA (0.3 μl) and each restriction enzyme (10 units) to a total volume of 30 μl. It was.
アガロースゲル（Ａｇａｒｏｓｅ ＬＥ、ナカライテスク）は、ＴＡＥバッファー（４．９８ｇ／ｌ Ｔｒｉｓ ｂａｓｅ（ナカライテスク）、１．１４２ｍｌ／ｌ氷酢酸（ナカライテスク）、１ｍＭ ＥＤＴＡ（ｐＨ８）（Ｄｏｊｉｎｄｏ））にて加熱溶解し、１％または２％となるように調製した。λＨｉｎｄIIIｄｉｇｅｓｔ（Ｔｏｙｏｂｏ）または１００ｂｐ ＤＮＡ Ｌａｄｄｅｒ（Ｔｏｙｏｂｏ）をＤＮＡサイズマーカーとして、ＤＮＡ試料は制限酵素に添付されている１０ｘサンプルバッファーを１／１０量と、ＤＭＳＯ（Ｓｉｇｍａ）にて１００倍希釈したＳＹＢＲ Ｇｒｅｅｎ Ｉ（Ｉｎｖｉｔｒｏｇｅｎ）を１／１０量加えたものを、ＴＡＥバッファーを用いて１００Vにて電気泳動を行い、Ｓａｆｅ Ｉｍａｇｅｒ（Ｉｎｖｉｔｒｏｇｅｎ）を用いて検出した。 Agarose gel (Agarose LE, Nacalai Tesque) was heated with TAE buffer (4.98 g / l Tris base (Nacalai Tesque), 1.142 ml / l glacial acetic acid (Nacalai Tesque), 1 mM EDTA (pH 8) (Dojindo)). Dissolved and prepared to 1% or 2%. λHindIII digest (Toyobo) or 100 bp DNA Ladder (Toyobo) is used as a DNA size marker, and a DNA sample is SYBR Green I diluted 100-fold with DMSO (Sigma) in 1/10 volume of 10 × sample buffer attached to a restriction enzyme. What added 1/10 amount of (Invitrogen) was electrophoresed at 100V using TAE buffer, and detected using Safe Imager (Invitrogen).
ゲルからのＤＮＡの回収にはＭａｇＥｘｔｒａｃｔｏｒ（Ｔｏｙｏｂｏ）を用い、操作はそのマニュアルに従って行った。詳細には、まずアガロースゲル電気泳動後Ｓａｆｅ Ｉｍａｇｅｒ上で目的のバンドをなるべく小さくなるようにメスで切り出し、吸着液を４００μｌ加えて室温に放置してゲルを完全に溶解させた。次に磁性ビーズを３０μｌ加えて時々撹拌しながら室温に２分放置した。ＤＮＡを吸着した磁性ビーズはマグネットスタンドを用いて吸着し、上清は捨てた。回収した磁気ビーズに洗浄液を６００μｌ加えてボルテックスミキサーで１０秒撹拌し、マグネットスタンドを用いてＤＮＡを吸着した磁性ビーズを同様の手法で回収した。これに７５％エタノールを１ｍｌ加えてボルテックスミキサーで１０秒撹拌し、ＤＮＡを吸着した磁性ビーズはマグネットスタンドを用いて回収した。これをスピンダウンして完全に上清を捨て、５５度で2分間乾燥させた後、水又はＴＥを２５〜１００μｌ加えて時々撹拌しながら、室温で２分間放置した。ＤＮＡを解離させた後の磁性ビーズはマグネットスタンドを用いて分離し、ＤＮＡを含む上清を回収した。 MagExtractor (Toyobo) was used for DNA recovery from the gel, and the operation was performed according to the manual. Specifically, first, after agarose gel electrophoresis, the target band was cut out with a scalpel so as to be as small as possible on the Safe Imager, and 400 μl of the adsorbed solution was added and left at room temperature to completely dissolve the gel. Next, 30 μl of magnetic beads were added and left at room temperature for 2 minutes with occasional stirring. The magnetic beads adsorbed with DNA were adsorbed using a magnet stand, and the supernatant was discarded. 600 μl of the washing solution was added to the collected magnetic beads and stirred for 10 seconds with a vortex mixer, and the magnetic beads adsorbed with DNA using a magnetic stand were collected by the same method. 1 ml of 75% ethanol was added thereto and stirred for 10 seconds with a vortex mixer, and the magnetic beads adsorbed with DNA were collected using a magnet stand. This was spun down, and the supernatant was completely discarded. After drying at 55 ° C. for 2 minutes, 25 to 100 μl of water or TE was added and allowed to stand at room temperature for 2 minutes with occasional stirring. The magnetic beads after the DNA was dissociated were separated using a magnet stand, and the supernatant containing the DNA was collected.
Ｌｉｇａｔｉｏｎ反応にはＤＮＡ Ｌｉｇａｔｉｏｎ Ｋｉｔ Ｖｅｒ．２（Ｔａｋａｒａ）を用い、操作はそのマニュアルに従って行った。詳細には、約２５ｆｍｏｌのプラスミドベクターおよび約２５〜２５０ｆｍｏｌのインサートＤＮＡの混合溶液に等量のＬｉｇａｔｉｏｎ Ｍｉｘを添加して混和した後、摂氏１６度で３０分間反応させた。 In the Ligation reaction, DNA Ligation Kit Ver. 2 (Takara) was used and the operation was performed according to the manual. Specifically, an equal volume of Ligation Mix was added to and mixed with a mixed solution of about 25 fmol of plasmid vector and about 25 to 250 fmol of insert DNA, and then reacted at 16 degrees Celsius for 30 minutes.
形質転換はＥ．ｃｏｌｉコンピテントセルＤＨ５α（Ｔａｋａｒａ）、またはＫＲＸ（Ｔａｋａｒａ）を用いて行った。詳細には、１００μｌのコンピテントセルを氷上にて溶解し、ＤＮＡ溶液１μｌまたはＬｉｇａｔｉｏｎ反応液１μｌを加えて氷上で３０分間放置した後、摂氏４２度で４５秒間加熱した。その後さらに氷上で５分間放置し、ＬＢ培地５００μｌを加えて摂氏３７度で１時間培養後、１００μｇ／ｍｌのアンピシリンまたは５０μｇ／ｍｌのカナマイシン（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ）を含む選択培地（ＬＢ培地）に植えて、摂氏３７度にて一晩培養した。翌日、コロニーを１００μｇ／ｍｌのアンピシリンまたは５０μｇ／ｍｌのカナマイシンを含む１〜５ｍｌの液体培地（ＬＢ培地）に植えつぎ、摂氏３７度にて１６時間培養した。 Transformation is performed in E. coli. E. coli competent cells DH5α (Takara) or KRX (Takara) were used. Specifically, 100 μl of competent cells were dissolved on ice, 1 μl of DNA solution or 1 μl of ligation reaction solution was added and left on ice for 30 minutes, and then heated at 42 degrees Celsius for 45 seconds. Thereafter, the mixture is further left on ice for 5 minutes, added with 500 μl of LB medium, cultured at 37 ° C. for 1 hour, and then planted in a selective medium (LB medium) containing 100 μg / ml ampicillin or 50 μg / ml kanamycin (Wako Chemicals). Incubate overnight at 37 degrees Celsius. The next day, the colonies were inoculated into 1 to 5 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin or 50 μg / ml kanamycin and cultured at 37 degrees Celsius for 16 hours.
大腸菌からのプラスミドの回収にはＭｉｎｉｐｒｅｐ Ｋｉｔ（Ｑｉａｇｅｎ）を用い、操作はそのマニュアルに従って行った。詳細には、まず５ｍｌの大腸菌培養液を約２，０００ｘｇ、１０分の遠心に付し、上清をデカンテーションまたはピペッティングで除去して大腸菌の沈殿を得た。この沈殿に氷冷したＲＮａｓｅ入りＰ１バッファーを２５０μｌ加えてサスペンドし、Ｐ２バッファーを２５０μｌ加えて室温に５分放置してアルカリＳＤＳにより菌体を破砕した。その後Ｎ３バッファーを３５０μｌ加えて中性化した。その菌体破砕液をｓｐｉｎ ｃｏｌｕｍｎに移し、約１３，２００ｘｇ、３０秒〜１分の遠心に付してプラスミドをカラムに吸着させた。カラム素通り液はデカンテーションにて除去した。次にカラムにＰＥバッファーを７５０μｌ加えて約１３，２００ｘｇ、３０秒〜１分の遠心に付してカラムを洗浄した。カラム素通り液はデカンテーションにて除去した。さらにバッファーを加えずにもう一度約１３，２００ｘｇ、３０秒〜１分の遠心に付してカラムに残った液滴を完全に除去した。カラムを新しい回収用マイクロチューブにとりつけ、カラムにＥＢバッファーを５０μｌ加えて約１３，２００ｘｇ、３０秒〜１分の遠心に付してカラムからプラスミドを溶出し回収した。 Miniprep Kit (Qiagen) was used to recover the plasmid from E. coli, and the operation was performed according to the manual. Specifically, first, 5 ml of E. coli culture solution was centrifuged at about 2,000 × g for 10 minutes, and the supernatant was removed by decantation or pipetting to obtain an E. coli precipitate. To this precipitate, 250 μl of ice-cooled RNase-containing P1 buffer was added and suspended, 250 μl of P2 buffer was added and left at room temperature for 5 minutes, and the cells were disrupted with alkaline SDS. Thereafter, 350 μl of N3 buffer was added for neutralization. The cell disruption solution was transferred to spin column and centrifuged at about 13,200 × g for 30 seconds to 1 minute to adsorb the plasmid to the column. The column passage liquid was removed by decantation. Next, 750 μl of PE buffer was added to the column, and the column was washed by centrifugation at about 13,200 × g for 30 seconds to 1 minute. The column passage liquid was removed by decantation. Further, the liquid droplets remaining on the column were completely removed by further centrifuging at about 13,200 × g for 30 seconds to 1 minute without adding any buffer. The column was attached to a new collection microtube, 50 μl of EB buffer was added to the column, and centrifuged at about 13,200 × g for 30 seconds to 1 minute to elute and collect the plasmid from the column.
ＬＢ液体培地
１０ｇ／ｌ Ｂａｃｔｏ−ｔｒｙｐｔｏｎｅ（ナカライテスク）、５ｇ／ｌ Ｂａｃｔｏ−ｙｅａｓｔ ｅｘｔｒａｃｔ（ナカライテスク）、５ｇ／ｌ ＮａＣｌ（ナカライテスク）、１ｇ／ｌ ｇｌｕｃｏｓｅ（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ）。オートクレーブにて滅菌して調製。
LB liquid medium 10 g / l Bacto-tryptone (Nacalai Tesque), 5 g / l Bacto-yeast extract (Nacalai Tesque), 5 g / l NaCl (Nacalai Tesque), 1 g / l glucose (Wako Chemicals). Sterilized in an autoclave.
ＬＢ寒天培地
１０ｇ／ｌ Ｂａｃｔｏ−ｔｒｙｐｔｏｎｅ（ナカライテスク）、５ｇ／ｌ Ｂａｃｔｏ−ｙｅａｓｔ ｅｘｔｒａｃｔ（ナカライテスク）、５ｇ／ｌ ＮａＣｌ（ナカライテスク）、１ｇ／ｌ ｇｌｕｃｏｓｅ（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ）、１５ｇ／ｌ Ａｇａｒ（ナカライテスク）。オートクレーブにて滅菌後、温度が４５度程度まで下がったところで抗生物質（１００μｇ／ｍｌのアンピシリンまたは５０μｇ／ｍｌのカナマイシン（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ））を加え、プラスチックディシュに流し込んで調製。
ＴＥ（ｐＨ８）（１０ｍＭ Ｔｒｉｓ−ＨＣｌ, １ｍＭ ＥＤＴＡ）（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ）
LB agar medium 10 g / l Bacto-tryptone (Nacalai Tesque), 5 g / l Bacto-yeast extract (Nacalai Tesque), 5 g / l NaCl (Nacalai Tesque), 1 g / l glucose (Wako Chemicals), 15 g / l Ala Tesque). After sterilization in an autoclave, when the temperature drops to about 45 degrees, antibiotics (100 μg / ml ampicillin or 50 μg / ml kanamycin (Wako Chemicals)) are added and poured into a plastic dish.
TE (pH 8) (10 mM Tris-HCl, 1 mM EDTA) (Wako Chemicals)
（Ａ−２）蛋白質の精製
Ｒ−ＣａＭＰ１．０７およびＲ−ＣａＭＰ１．０１の蛋白質精製にはこれらの蛋白質がＨｉｓタグを持っていることを利用して、Ｈｉｓタグに特異的に結合するＮｉ−ＮＴＡ ａｇａｒｏｓｅ（Ｑｉａｇｅｎ）を用い、操作はそのマニュアルに従って行った。詳細には、ｐＲＳＥＴＢ−Ｒ−ＣａＭＰ１．０７をＥ．ｃｏｌｉコンピテントセルＫＲＸに形質転換し、１００μｇ／ｍｌのアンピシリンを含むＬＢ選択培地に植え、摂氏３７度で一晩培養した。コロニーを１００μｇ／ｍｌのアンピシリンを含む１０ｍｌの液体培地（ＬＢ培地）に植えつぎ、摂氏３７度にて１６時間培養した。培養液１０ｍｌをさらに１００μｇ／ｍｌのアンピシリンを含む２００ｍｌの液体培地（ＬＢ培地）に植えつぎ、吸光度ＯＤ６００で０．５〜１となるまで摂氏３７度で培養した後、最終濃度が１％になるようにラムノース（プロメガ）を加えて、摂氏１８〜２２．５度で４〜５時間さらに培養した。
(A-2) Protein purification The protein purification of R-CaMP1.07 and R-CaMP1.01 utilizes the fact that these proteins have His tags, and Ni- that specifically binds to His tags. The operation was performed according to the manual using NTA agarose (Qiagen). Specifically, pRSET B -R-CaMP 1.07 E. coli competent cells KRX were transformed into LB selection medium containing 100 μg / ml ampicillin and cultured overnight at 37 degrees Celsius. The colony was inoculated into 10 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin and cultured at 37 degrees Celsius for 16 hours. 10 ml of the culture solution is further planted in 200 ml of liquid medium (LB medium) containing 100 μg / ml ampicillin and cultured at 37 degrees Celsius until the absorbance OD600 is 0.5 to 1, after which the final concentration becomes 1%. Rhamnose (Promega) was added and cultured at 18-22.5 degrees Celsius for 4-5 hours.
３，０００回転１５分遠心して（６２００遠心機、Ｋｕｂｏｔａ）、大腸菌を回収した。１ｍｌのＬＢ培地で大腸菌を懸濁した。摂氏−２０度で３０分凍らせたのち、室温で３０分解凍した。もう１度凍結、解凍を繰り返した。氷上で冷やした４０ｍｌのｓｕｓｐｅｎｓｉｏｎ ｂｕｆｆｅｒ（２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８）（Ｓｉｇｍａ）、１ｍＭ ２−メルカプトエタノール（ナカライテスク）、蛋白分解酵素阻害剤（０．１ｍＭ ＰＭＳＦ、５μｇ／ｍｌ ロイペプチン（Ｗａｋｏ Ｃｈｅｍｉｃａｌｓ））を加え、よく混ぜて大腸菌を懸濁した。摂氏４度にて１００,０００ｘｇで１５分間遠心し、上清を得た。５Ｍ ＮａＣｌを最終濃度が０．３Ｍとなるように加え、２ｍｌの５０％ Ｎｉ−ＮＴＡ ａｇａｒｏｓｅ（Ｑｉａｇｅｎ；蛋白質結合能５〜１０ｍｇ／ｍｌレジン）をさらに加えて１時間室温でおだやかに混ぜて反応させた。反応液を空のカラム（エコノカラム；カラムサイズ 〜２０ｍｌ（Ｂｉｏ−Ｒａｄ））に移し、余分の液がカラムから滴下してなくなるのを待った。１０ｍｌの洗浄液（５０ｍＭ ＮａＨ２ＰＯ４（ｐＨ８）（ナカライテスク）、０．３Ｍ ＮａＣｌ、２０ｍＭ ｉｍｉｄａｚｏｌｅ（ナカライテスク））で2回洗浄した後、３〜４ｍｌの回収液（５０ｍＭ ＮａＨ２ＰＯ４（ｐＨ８）（ナカライテスク）、０．３Ｍ ＮａＣｌ、２５０ｍＭ ｉｍｉｄａｚｏｌｅ（ナカライテスク））にて溶出し、Ｈｉｓタグ付きの蛋白質をカラムから回収した。次に、回収した液を透析チューブ（Ｓａｎｋｏｕｊｕｎｙａｋｕ）に入れて１２５ｍｌまたはそれ以上のＫＭバッファー（０．１Ｍ ＫＣｌ（ナカライテスク）、２０ｍＭ ＭＯＰＳ−Ｔｒｉｓ（ｐＨ７．５）（Ｄｏｊｉｎｄｏ））で摂氏４度にて透析した。ＫＭバッファーは４〜５時間ごとに交換し、液交換を３回以上行った後、透析チューブから蛋白質の溶液を回収した。 After centrifugation at 3,000 rpm for 15 minutes (6200 centrifuge, Kubota), E. coli was recovered. Escherichia coli was suspended in 1 ml of LB medium. After freezing at -20 degrees Celsius for 30 minutes, it was thawed at room temperature for 30 minutes. Freezing and thawing were repeated once more. 40 ml suspension buffer (25 mM Tris-HCl (pH 8) (Sigma), 1 mM 2-mercaptoethanol (Nacalai Tesque)), protease inhibitor (0.1 mM PMSF, 5 μg / ml leupeptin (Wako Chemicals)) chilled on ice Suspended Escherichia coli at 4 degrees Celsius and centrifuged at 100,000 xg for 15 minutes to obtain a supernatant, 5M NaCl was added to a final concentration of 0.3M, and 2ml of 50ml was added. % Ni-NTA agarose (Qiagen; protein binding ability 5 to 10 mg / ml resin) was further added, and the mixture was gently mixed at room temperature for 1 hour to allow the reaction to proceed, and the reaction solution was added to an empty column (Econo column; column size ˜20 ml (Bio- Rad)) and the excess liquid is not dripped from the column. After washing twice with 10 ml of washing solution (50 mM NaH 2 PO 4 (pH 8) (Nacalai Tesque), 0.3 M NaCl, 20 mM imidazole (Nacalai Tesque)), 3 to 4 ml of recovery solution (50 mM The His-tagged protein was recovered from the column by elution with NaH 2 PO 4 (pH 8) (Nacalai Tesque), 0.3 M NaCl, 250 mM imidazole (Nacalai Tesque). In sanko junyaku), it was dialyzed with 125 ml or more of KM buffer (0.1 M KCl (Nacalai Tesque), 20 mM MOPS-Tris (pH 7.5) (Dojindo)) at 4 degrees Celsius. After changing every hour, and after changing liquids 3 times or more It was recovered solution of the protein from the dialysis tube.
蛋白質の濃度測定にはプロテインアッセイキット（Ｂｉｏ−Ｒａｄ）を用い、操作はそのマニュアルに従ってＢｒａｄｆｏｒｄ法（Bradford, M. M. Anal. Biochem. 1976, 72, 248―254.）で測定した。まず、１０〜２００μｇ／ｍｌとなるように水で希釈した蛋白質の溶液５０μｌにＢｒａｄｆｏｒｄ試薬を１ｍｌ加えて３０分後に５９５ｎｍの吸光度を測定した。蛋白質の基準濃度は牛血清アルブミンを基準蛋白質として用いて測定して求めた。測定は室温にて行った。 The protein concentration was measured using a protein assay kit (Bio-Rad), and the operation was measured by the Bradford method (Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.) According to the manual. First, 1 ml of Bradford reagent was added to 50 μl of a protein solution diluted with water to 10 to 200 μg / ml, and the absorbance at 595 nm was measured 30 minutes later. The reference concentration of the protein was determined by measuring using bovine serum albumin as the reference protein. The measurement was performed at room temperature.
（Ｂ）Ｒ−ＣａＭＰ１．０７を用いた測定法
（Ｂ−１）カルシウム結合能の測定
Ｒ−ＣａＭＰ１．０７蛋白質のカルシウム結合能はさまざまなカルシウム濃度溶液中における蛍光強度を測定して得られたカルシウム濃度―蛍光強度の容量反応曲線に基づいて算出した。既述のように精製したＲ−ＣａＭＰ１．０７蛋白質はＫＭバッファーで最終濃度が０．３μＭとなるように希釈した。蛍光強度測定には、蛍光分光光度計Ｆ−２５００（Ｈｉｔａｃｈｉ）を用い５６０ｎｍで励起し５８５ｎｍで蛍光を記録した。まず測定標品に２０ｍＭ ＢＡＰＴＡ（（Ｄｏｊｉｎｄｏ））を添加してカルシウム非存在下における測定を行った後、逐次ＣａＣｌ２をさまざまな濃度になるように添加して測定した。測定は室温にて行った。
(B) Measurement method using R-CaMP1.07 (B-1) Measurement of calcium binding ability The calcium binding ability of R-CaMP1.07 protein was obtained by measuring the fluorescence intensity in various calcium concentration solutions. It was calculated based on a volumetric response curve of calcium concentration-fluorescence intensity. The R-CaMP1.07 protein purified as described above was diluted with KM buffer to a final concentration of 0.3 μM. For fluorescence intensity measurement, a fluorescence spectrophotometer F-2500 (Hitachi) was used to excite at 560 nm and record fluorescence at 585 nm. First, 20 mM BAPTA ((Dojindo)) was added to the measurement sample and measurement was performed in the absence of calcium, and then CaCl 2 was successively added at various concentrations and measured. The measurement was performed at room temperature.
（Ｂ−２）ＨｅＬａ細胞の培養とプラスミドの導入
炭酸ガス培養器を用いて、培地（ＤＭＥＭ（Ｇｉｂｃｏ）、１０％ Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（Ｇｉｂｃｏ）、１ｘペニシリン・ストレプトマイシン（Ｇｉｂｃｏ））にてＨｅＬａ細胞を摂氏３７度で培養し、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ２０００（Ｉｎｖｉｔｒｏｇｅｎ）を用いて培養細胞にｐＮ１−Ｒ−ＣａＭＰ１．０７のプラスミドを導入した。導入操作は試薬のマニュアルに従って行った。まず、血清を含まないＤＭＥＭ５０μｌでプラスミド０．８μｇを希釈した。次に２μｌのＬｉｐｏｆｅｃｔａｍｉｎｅ ２０００を血清を含まないＤＭＥＭ５０μｌに加え室温で５分放置した。その後両希釈液を混合して室温で２０分放置した。この混合液の全量を２４穴培養シャーレ中のＨｅＬａ細胞に投与してプラスミドを導入した。プラスミドを導入した後細胞は摂氏３７度で１〜３日培養した。
(B-2) HeLa cell culture and plasmid introduction Using a carbon dioxide incubator, HeLa cells were cultured in a medium (DMEM (Gibco), 10% Fetal Bovine Serum (Gibco), 1x penicillin streptomycin (Gibco)). After culturing at 37 degrees Celsius, the plasmid pN1-R-CaMP1.07 was introduced into the cultured cells using Lipofectamine 2000 (Invitrogen). The introduction operation was performed according to the reagent manual. First, 0.8 μg of plasmid was diluted with 50 μl of DMEM without serum. Next, 2 μl of Lipofectamine 2000 was added to 50 μl of serum-free DMEM and left at room temperature for 5 minutes. Thereafter, both dilutions were mixed and allowed to stand at room temperature for 20 minutes. The entire amount of this mixed solution was administered to HeLa cells in a 24-well culture dish to introduce the plasmid. After introducing the plasmid, the cells were cultured at 37 degrees Celsius for 1-3 days.
（Ｂ−３）ＨｅＬａ細胞での蛍光測定
蛍光測定にはコンピューターにて制御（アクアコスモス（浜松ホトニクス））されたＣＣＤカメラ（ＯＲＣＡ−ＥＲ、浜松ホトニクス）を搭載した倒立蛍光顕微鏡（ＩＸ７０（オリンパス）、ＷＩＹフィルターセット（オリンパス）、対物レンズ２０ｘ（オリンパス））を用いた。プラスミドを導入した細胞を顕微鏡にセットし、ＨＢＳバッファー（１０７ｍＭ NａＣｌ、６ｍＭ ＫＣｌ、１．２ｍＭ ＭｇＳＯ４（ナカライテスク）、２ｍＭ ＣａＣｌ２、１．２ｍＭ ＫＨ２ＰＯ４（ナカライテスク）、１１．５ｍＭ ｇｌｕｃｏｓｅ、２０ｍＭ ＨＥＰＥＳ（Ｄｏｊｉｎｄｏ）（ｐＨ７．４））を細胞外液として還流し、１００μＭ ＡＴＰ（Ｓｉｇｍａ）を細胞外に投与して細胞を刺激し、その際に起こる細胞内カルシウム濃度変化を蛍光強度変化として検出した。測定は室温にて行った。
(B-3) Fluorescence measurement with HeLa cells An inverted fluorescence microscope (IX70 (Olympus)) equipped with a CCD camera (ORCA-ER, Hamamatsu Photonics) controlled by a computer (Aquacosmos (Hamamatsu Photonics)) for fluorescence measurement , WIY filter set (Olympus), objective lens 20x (Olympus)). Cells into which the plasmid was introduced were set in a microscope, and HBS buffer (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO 4 (Nacalai Tesque), 2 mM CaCl 2 , 1.2 mM KH 2 PO 4 (Nacalai Tesque)), 11.5 mM glucose , 20 mM HEPES (Dojindo) (pH 7.4)) was refluxed as an extracellular solution, and 100 μM ATP (Sigma) was administered extracellularly to stimulate the cells. Detected as. The measurement was performed at room temperature.
本発明は、筋肉の収縮、神経興奮性やホルモン分泌、酵素活性の変化などの各種の細胞機能の調節因子として、生体機能の維持および調節に不可欠な役割を担っているカルシウム濃度の生体内での変動を、従来のものに比べ極めて高感度で測定するカルシウムセンサー蛋白質を提供するもので、その利用価値は高く、生体機序の解明や医学・創薬といった分野に大きく貢献するものである。 The present invention provides a calcium concentration that plays an essential role in the maintenance and regulation of biological functions as a regulator of various cellular functions such as muscle contraction, nerve excitability, hormone secretion, and changes in enzyme activity. Provides a calcium sensor protein that can measure the fluctuations of the protein with extremely high sensitivity compared to the conventional one, and its use value is high, and it greatly contributes to the field of elucidation of biological mechanisms, medicine and drug discovery.
Claims (10)
（ａ）配列番号１で示されるアミノ酸配列；
（ｂ）３つのアミノ酸からなる配列 Ｍｅｔ−Ｘａａ１−Ｘａａ２（ここでＸａａ１及びＸａａ２はそれぞれ独立して任意のアミノ酸である）（リンカーＡ）（配列番号２）；
（ｃ）ミオシン軽鎖キナーゼ蛋白質、又はカルモジュリン結合部位を含むその部分アミノ酸配列；
（ｄ）前記（ｃ）の配列と後記（ｅ）の配列とを連結する、Ｉｓｏ−Ｉｓｏ、Ｉｓｏ−Ｌｅｕ、Ｉｓｏ−Ｍｅｔ、Ｉｓｏ−Ｐｒｏ、Ｉｓｏ−Ｖａｌ、Ｉｓｏ−Ｇｌｙ、Ｉｓｏ−Ａｌａ、Ｌｅｕ−Ｉｓｏ、Ｌｅｕ−Ｌｅｕ、Ｌｅｕ−Ｍｅｔ、Ｌｅｕ−Ｐｒｏ、Ｌｅｕ−Ｖａｌ、Ｌｅｕ−Ｇｌｙ、Ｌｅｕ−Ａｌａ、Ｍｅｔ−Ｉｓｏ、Ｍｅｔ−Ｌｅｕ、Ｍｅｔ−Ｍｅｔ、Ｍｅｔ−Ｐｒｏ、Ｍｅｔ−Ｖａｌ、Ｍｅｔ−Ｇｌｙ、Ｍｅｔ−Ａｌａ、Ｐｒｏ−Ｉｓｏ、Ｐｒｏ−Ｌｅｕ、Ｐｒｏ−Ｍｅｔ、Ｐｒｏ−Ｐｒｏ、Ｐｒｏ−Ｖａｌ、Ｐｒｏ−Ｇｌｙ、Ｐｒｏ−Ａｌａ、Ｖａｌ−Ｉｓｏ、Ｖａｌ−Ｌｅｕ、Ｖａｌ−Ｍｅｔ、Ｖａｌ−Ｐｒｏ、Ｖａｌ−Ｖａｌ、Ｖａｌ−Ｇｌｙ、Ｖａｌ−Ａｌａ、Ｇｌｙ−Ｉｓｏ、Ｇｌｙ−Ｌｅｕ、Ｇｌｙ−Ｍｅｔ、Ｇｌｙ−Ｐｒｏ、Ｇｌｙ−Ｖａｌ、Ｇｌｙ−Ｇｌｙ、Ｇｌｙ−Ａｌａ、Ａｌａ−Ｉｓｏ、Ａｌａ−Ｌｅｕ、Ａｌａ−Ｍｅｔ、Ａｌａ−Ｐｒｏ、Ａｌａ−Ｖａｌ、Ａｌａ−Ｇｌｙ及びＡｌａ−Ａｌａからなる群より選択される何れか一のアミノ酸配列（リンカーＢ）；
（ｅ）配列番号３で示される配列のＸ番目〜２３６番目までのアミノ酸配列であって、１５１番目及び／又は１５２番目及び／又は１６９番目及び／又は１７１番目及び／又は２１９番目のアミノ酸を他のアミノ酸に置換したもの（ここで、Ｘは１５１〜１５３の任意の位置である）；
（ｆ）前記（ｅ）の配列と後記（ｇ）の配列を連結する、６つのアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｘａａ５−Ｇｌｙ−Ｇｌｙ−Ｘａａ６（ここでＸａａ５及びＸａａ６はそれぞれ独立して任意のアミノ酸である）（配列番号４）；
（ｇ）配列番号３で示される配列の１番目〜Ｙ番目までのアミノ酸配列であって、
１番目及び／又は８番目及び／又は５２番目及び／又は５４番目及び／又は７６番目及び／又は１３６番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列（ここで、Ｙは１４４〜１５０の任意の位置である）；
（ｈ）前記（ｇ）の配列と後記（ｉ）の配列とを連結するアミノ酸配列Ｔｈｒ−Ａｒｇ、Ｐｈｅ−Ａｒｇ、Ｔｒｐ−Ａｒｇ、Ｔｙｒ−Ａｒｇ、Ｇｌｙ−Ａｒｇ、Ａｌａ−Ａｒｇ又はＴｈｒ（リンカーＣ）；
（ｉ）配列番号９で示される配列の２番目〜１４８番目までのアミノ酸配列であって、６３番目及び／又は７７番目及び／又は１０１番目及び／又は１１１番目及び／又は１２７番目のアミノ酸を他のアミノ酸に置換したアミノ酸配列；
（ｊ）前記（ｉ）の配列と後記（ｋ）の配列とを連結する、１５個のアミノ酸配列からなる配列Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ７−Ｇｌｙ−Ｇｌｙ−Ｘａａ８−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｇｌｙ−Ｘａａ９−Ｘａａ１０（ここでＸａａ７、Ｘａａ８、Ｘａａ９及びＸａａ１０はそれぞれ独立して任意のアミノ酸である）（リンカーＤ）（配列番号１０）；
（ｋ）Ｐｒｏ−Ｖａｌ−Ｌｙｓ−Ｇｌｎ−Ｔｈｒ−Ｌｅｕ−Ａｓｎ−Ｐｈｅ−Ａｓｐ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１２）、Ｇｌｎ−Ｃｙｓ−Ｔｈｒ−Ａｓｎ−Ｔｙｒ−Ａｌａ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｌｅｕ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｓｅｒ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１３）、Ｇｌｕ−Ｇｌｙ−Ａｒｇ−Ｇｌｙ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｔｈｒ−Ｃｙｓ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１４）又はＡｌａ−Ｔｈｒ−Ａｓｎ−Ｐｈｅ−Ｓｅｒ−Ｌｅｕ−Ｌｅｕ−Ｌｙｓ−Ｇｌｎ−Ａｌａ−Ｇｌｙ−Ａｓｐ−Ｖａｌ−Ｇｌｕ−Ｇｌｕ−Ａｓｎ−Ｐｒｏ−Ｇｌｙ（配列番号１５）のいずれかのアミノ酸配列。 Calcium sensor protein having the following amino acid sequences (a) to (k) in order from the N-terminus:
(A) the amino acid sequence represented by SEQ ID NO: 1;
(B) a sequence consisting of three amino acids Met-Xaa1-Xaa2 (where Xaa1 and Xaa2 are each independently any amino acid) (linker A) (SEQ ID NO: 2);
(C) a myosin light chain kinase protein, or a partial amino acid sequence thereof comprising a calmodulin binding site;
(D) The sequence of (c) and the sequence of (e) described below are linked, Iso-Iso, Iso-Leu, Iso-Met, Iso-Pro, Iso-Val, Iso-Gly, Iso-Ala, Leu -Iso, Leu-Leu, Leu-Met, Leu-Pro, Leu-Val, Leu-Gly, Leu-Ala, Met-Iso, Met-Leu, Met-Met, Met-Pro, Met-Val, Met-Gly , Met-Ala, Pro-Iso, Pro-Leu, Pro-Met, Pro-Pro, Pro-Val, Pro-Gly, Pro-Ala, Val-Iso, Val-Leu, Val-Met, Val-Pro, Val -Val, Val-Gly, Val-Ala, Gly-Iso, Gly-Leu, Gly-Met, Gl Any selected from the group consisting of Pro, Gly-Val, Gly-Gly, Gly-Ala, Ala-Iso, Ala-Leu, Ala-Met, Ala-Pro, Ala-Val, Ala-Gly and Ala-Ala Any one amino acid sequence (linker B);
(E) the amino acid sequence from the Xth to the 236th amino acid sequence of the sequence shown in SEQ ID NO: 3, except for the 151st and / or 152th and / or 169th and / or 171st and / or 219th amino acids (Wherein X is an arbitrary position of 151 to 153);
(F) A sequence Gly-Gly-Xaa5-Gly-Gly-Xaa6 consisting of six amino acid sequences connecting the sequence of (e) and the sequence of (g) to be described later (where Xaa5 and Xaa6 are each independently arbitrary (SEQ ID NO: 4);
(G) an amino acid sequence from the first to the Y-th of the sequence represented by SEQ ID NO: 3,
Amino acid sequence in which the first and / or eighth and / or 52 and / or 54 and / or 76 and / or 136 amino acids are substituted with other amino acids (where Y is any of 144 to 150) Position);
(H) Amino acid sequence Thr-Arg, Phe-Arg, Trp-Arg, Tyr-Arg, Gly-Arg, Ala-Arg or Thr (linker C) linking the sequence of (g) and the sequence of (i) described later );
(I) Amino acid sequence from 2nd to 148th of the sequence shown in SEQ ID NO: 9, except for the 63rd and / or 77th and / or 101st and / or 111th and / or 127th amino acids An amino acid sequence substituted with the amino acid of
(J) A sequence consisting of 15 amino acid sequences Gly-Gly-Gly-Xaa7-Gly-Gly-Xaa8-Gly-Gly-Gly-Gly, linking the sequence of (i) and the sequence of (k) described later -Gly-Gly-Xaa9-Xaa10 (where Xaa7, Xaa8, Xaa9 and Xaa10 are each independently any amino acid) (linker D) (SEQ ID NO: 10);
(K) Pro-Val-Lys-Gln-Thr-Leu-Asn-Phe-Asp-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 12 ), Gln-Cys-Thr-Asn-Tyr-Ala-Leu-Leu-Lys-Leu-Ala-Gly-Asp-Val-Glu-Ser-Asn-Pro-Gly (SEQ ID NO: 13), Glu-Gly-Arg -Gly-Ser-Leu-Leu-Thr-Cys-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly (SEQ ID NO: 14) or Ala-Thr-Asn-Phe-Ser-Leu-Leu-Lys- Gln-Ala-Gly-Asp-Val-Glu-Glu-Asn-Pro-Gly Any of the amino acid sequences of the number 15).
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