Development of sensors for monitoring intracellular ATP and GTP.

为了真实的实时监测细胞内ATP的动态变化，我们利用大肠杆菌腺苷酸激酶（AK）构建了一种基于FRET（fluorescence resonance energy transfer）的ATP传感器。首先，将AK基因插入pSE380表达载体的trc启动子下游，构建了野生型大肠杆菌AK的表达载体。用构建的载体转化大肠杆菌JM 109菌株，并分析过量产生的大肠杆菌AK蛋白。然后，使用错配引物和野生型AK表达载体作为模板，通过PCR将突变引入LID结构域的选定的三个位置，并确认其蛋白质的表达。将突变型AK表达载体导入大肠杆菌中， ...更多信息 转化到大肠杆菌CV2菌株中。CV2是一株温度敏感的菌株，在42℃下不能存活，如果突变蛋白不具有任何腺苷酸激酶活性，则转化AK基因的细菌不能在42℃下生长。所有突变株在42℃下均能存活，说明突变株具有磷酰基转移活性。其次，我们建立了AK突变体蛋白的纯化方法。利用Blue Sepharose和DE 53柱的组合可以纯化野生型和突变体蛋白。最后，将这些突变蛋白的腺苷酸激酶活性与野生型进行比较。其中两个突变体的活性约为野生型的70%，另一个突变体的活性约为野生型的30%，这表明所有突变体的蛋白质结构与野生型几乎相同，并且它们都能与ATP结合。在不久的将来，我们将得到有效性的答案。少

For real time monitoring of intracellular ATP dynamics, we have tried to develop FRET (fluorescence resonance energy transfer) based ATP sensor utilizing Escherichia coli adenylate kinase (AK). To make such a sensor, we planned to change one amino acid of LID domain, which moves largely when ATP binds to substrate binding region, to tryptophan as a donner of energy tansfer and modify cystein residue with a fluorophore as an acceptor.First of all, an expression vector of wild type E.coli AK was constructed by inserting AK gene downstream of trc promoter of pSE380 expression vector. E.coli JM109 strain was transformed with the constructed vector, and overproduced E.coli AK protein was analyzed. Then, mutations were introduced into selected three positions of LID domain by PCR using mismatch primers and wild type AK expression vector as a template, and expression of their proteins was confirmed. Enzyme activites of these AK mutants were first assesed by introducing mutant AK expression ve … More ctors into E.coli CV2 strain. CV2 is a temperature-sensitive strain and cannot survive at 42℃ without supply of active AK protein, so, if mutant protein does not have any adenylate kinase activity, bacteria transformed the AK gene cannot grow at 42℃. The results that all transformants with mutated AK genes could survive at 42℃ suggest that each AK mutant has phosphoryl transfer activity. Next, we established purification method of AK mutant proteins. Wild type and mutant proteins could be purified with combination of Blue sepharose and DE53 columns. Finally, adenylate kinase activities of these mutatnt proteins were compared with that of wild type. Two mutants had about 70% activities of wild type AK protein, and the other mutant had about 30% activity of wild type, suggesting that all mutant proteins have nearly the same structure as wild type protein and they can associate with ATP.Now we are performing to modify cystein residue of these three AK mutants with thiol reactive fluorophore and analyse change of fluorescence spectrum. In near future we will get the answer of validity. Less