In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human an...In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.展开更多
旨在探究丝氨酸苏氨酸蛋白激酶(serine/threonine protein kinase,STPK)基因在棉纤维发育中的功能,解析棉纤维细胞的分化和发育机理。从陆地棉遗传标准系TM-1中克隆基因GhD6PKL2,并对其序列及结构特征进行生物信息学分析、表达量分析及...旨在探究丝氨酸苏氨酸蛋白激酶(serine/threonine protein kinase,STPK)基因在棉纤维发育中的功能,解析棉纤维细胞的分化和发育机理。从陆地棉遗传标准系TM-1中克隆基因GhD6PKL2,并对其序列及结构特征进行生物信息学分析、表达量分析及过表达拟南芥的表型观察。GhD6PKL2含有典型的STPK保守序列位点。预测编码的蛋白相对分子质量为49.74 kD,等电点为6.17,含有多个丝氨酸苏氨酸磷酸化位点。表达模式结果表明,GhD6PKL2在与棉花纤维伸长阶段相吻合的、开花后20 d显著高表达。软件预测及在烟草叶片中的荧光蛋白定位结果均显示,GhD6PKL2编码的蛋白质定位在细胞膜上。诱饵自激活试验验证GhD6PKL2没有自激活活性及毒性。过表达拟南芥,能使转基因拟南芥表现出表皮毛数量增多,同时主根变短、侧根数目增多的表型。表明该丝氨酸苏氨酸蛋白激酶基因在拟南芥表皮毛及主侧根发育方面发挥一定作用,可能为一个棉纤维伸长发育阶段的潜在调控基因。展开更多
基金Supported by the National Natural Science Foundation of China(No.30970608)the Applicative Technological Project of Bureau of Science and Technology of Changchun City, China(No.2009045)+1 种基金the Development and Planning Major Program of Jilin Provincial Science and Technology Department, China(No.20100948)the Innovation Method Fund of China (No.2008IM040800)
文摘In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.
文摘旨在探究丝氨酸苏氨酸蛋白激酶(serine/threonine protein kinase,STPK)基因在棉纤维发育中的功能,解析棉纤维细胞的分化和发育机理。从陆地棉遗传标准系TM-1中克隆基因GhD6PKL2,并对其序列及结构特征进行生物信息学分析、表达量分析及过表达拟南芥的表型观察。GhD6PKL2含有典型的STPK保守序列位点。预测编码的蛋白相对分子质量为49.74 kD,等电点为6.17,含有多个丝氨酸苏氨酸磷酸化位点。表达模式结果表明,GhD6PKL2在与棉花纤维伸长阶段相吻合的、开花后20 d显著高表达。软件预测及在烟草叶片中的荧光蛋白定位结果均显示,GhD6PKL2编码的蛋白质定位在细胞膜上。诱饵自激活试验验证GhD6PKL2没有自激活活性及毒性。过表达拟南芥,能使转基因拟南芥表现出表皮毛数量增多,同时主根变短、侧根数目增多的表型。表明该丝氨酸苏氨酸蛋白激酶基因在拟南芥表皮毛及主侧根发育方面发挥一定作用,可能为一个棉纤维伸长发育阶段的潜在调控基因。