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海带核基因组编码CbbX蛋白的原核表达与纯化

DOI: 10.7668/hbnxb.2015.02.013, PP. 64-71

Keywords: 海带,核基因组,CbbX,蛋白,表达与纯化

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Abstract:

为研究海带核基因组编码的nucCbbX蛋白的结构和功能,根据海带配子体核基因cbbX(GenBank登录号:NP_053838)设计含有酶切位点的引物,通过RT-PCR方法获得末端连接NdeⅠ、XhoⅠ酶切位点的完整ORF。序列保守性分析表明,预测的海带成熟nucCbbX蛋白序列含具有AAA+结构域、Walker-A和Walker-BATP结合位点及ATP水解酶和RuBisCo活化酶活性相关位点。序列相似度分析表明,海带nucCbbX与其质体基因组编码的ptCbbX和类球红细菌的RsCbbX蛋白氨基酸序列相似度均较低,分别为37.4%和36.8%,相比ptCbbX与RsCbbX相似度较高为57.6%。以RsCbbX蛋白单体晶体结构模型为模板,预测获得的海带nucCbbX蛋白三级结构中N端为一个α/β子域含有5个α螺旋和5个β折叠,C端为α螺旋子域含有5个α螺旋。为进一步研究海带nucCbbX蛋白晶体结构,通过双酶切和连接反应成功构建pET28a-cbbX原核表达载体,并转化至BL21感受态细胞,获得pET28a-cbbX/BL21重组菌株。利用终浓度为1%的IPTG进行诱导后,重组蛋白主要以包涵体形式存在,分子量为50.2kDa,较nucCbbX成熟蛋白(44.7kDa)大。重组蛋白经变性、纯化和复性后用于蛋白结晶,初步获得海带nucCbbX蛋白晶体。

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