Development of sensors for monitoring intracellular ATP and GTP.

点击翻译按钮获取中文摘要

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