脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

Expression of Calmodulin binding Domain of Neuronal Nitric Synthase and Its Binding Activity to Calmodulin

用ＰＣＲ法克隆出ｎＮＯＳ的ＣａＭ结合区基因（ｎＮＯＳ２４５５～２９８８ｂｐ），并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为９０％以上的重组蛋白，分子量为２２ｋＤａ，ＣａＭＯｖｅｒｌａｙａｓａｙ证实该蛋白具有ＣａＭ的结合活性。由于所表达的重组蛋白既具有序列特异性又具有ＣａＭ的结合活性，因此，可将它作为筛选ｎＮＯＳ特异性抑制肽的靶蛋白，亦可用于特异性抗体的制备。

To facilitate to study the associations of nNOS functions with its calmodulin(CaM) binding domain and to prepare nNOS specific inhibiting peptides from phage peptide library,we have amplified the coding gene of nNOS CaM binding domain(nNOS2544 2988bp) and expressed it in E.coli .The recombinant product in size of 22kDa was purified (over 90% in pure) by His.Tag Sephorose column and its obvious CaM binding activity was detected with CaM overlay assay.Since the sequence specificity and effective calmodulin binding activity,the protein was considered as an ideal target for screening nNOS specific peptides from peptide library and also an antigen for raising nNOS antibody.