蛋白质Tat转运系统不同于细菌中普遍存在的Sec转运系统 ,而与植物叶绿体中蛋白质转运的ΔpH依赖系统相似 .通过Tat系统转运的蛋白质底物含有特征性的双精氨酸保守序列核心S/T R R x F L K的信号肽 ,其h区的疏水性低 ,c区有由高赖氨酸、...蛋白质Tat转运系统不同于细菌中普遍存在的Sec转运系统 ,而与植物叶绿体中蛋白质转运的ΔpH依赖系统相似 .通过Tat系统转运的蛋白质底物含有特征性的双精氨酸保守序列核心S/T R R x F L K的信号肽 ,其h区的疏水性低 ,c区有由高赖氨酸、高精氨酸构成的避开Sec系统信号 ,信号肽和成熟蛋白质的组成对蛋白质的转运都有影响 .TatA、TatB、TatC和TatE四种蛋白质参与了大肠杆菌的Tat转运系统 .被转运的底物蛋白质绝大多数为与细菌厌氧呼吸有关的含氧化还原辅因子的酶 ,并以折叠形式转运 .展开更多
为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and lig...为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中,成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达,通过改变培养温度和IPTG浓度等条件,得到了能够大量表达目标蛋白的重组子。结果表明:大肠杆菌BL21(DE3)是gsiB基因表达的最佳宿主菌;18℃低温诱导培养有利于gsiB基因的大量表达;0.1mmol/LIPTG足够诱导gsiB基因表达,增加IPTG浓度(0.1mmol/L～1.0mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达,其分子量大小与预期相符。展开更多
Background: CACNA1A encodes Cav2.1, the pore- forming subunit of P/Q- type voltage- gated calcium channel complexes. Mutations in CACNA1A cause a wide ran ge of neurologic disturbances variably associated with cerebel...Background: CACNA1A encodes Cav2.1, the pore- forming subunit of P/Q- type voltage- gated calcium channel complexes. Mutations in CACNA1A cause a wide ran ge of neurologic disturbances variably associated with cerebellar degeneration. Functional studies to date focus on electrophysiologic defects that do not adequ ately explain the phenotypic findings. Objective: To investigate whether some mi ssense mutations might interfere with protein folding and trafficking, eventuall y leading to protein aggregation and neuronal injury. Methods: The authors studi ed the functional consequences of two pore missense mutations, C287Y and G293R, in two families with EA2, one newly discovered and the other previously reported . Both mutations caused episodic and interictal ataxia. The biophysical properti es of mutant and wild type calcium channels were examined by whole- cell patch - clamp recordings in transfected COS- 7 cells. The plasma membrane targeting was visualized by confocal fluorescence imaging on Cav2.1 tagged with green fluo rescent protein. Results: The mutant channels exhibited a marked reduction in cu rrent expression and deficiencies in plasma membrane targeting. Conclusions: In addition to altered channel function, the deficiency in protein misfolding and t rafficking associated with the C287Y and G293R mutants may contribute to the slo wly progressive cerebellar ataxia.展开更多
文摘蛋白质Tat转运系统不同于细菌中普遍存在的Sec转运系统 ,而与植物叶绿体中蛋白质转运的ΔpH依赖系统相似 .通过Tat系统转运的蛋白质底物含有特征性的双精氨酸保守序列核心S/T R R x F L K的信号肽 ,其h区的疏水性低 ,c区有由高赖氨酸、高精氨酸构成的避开Sec系统信号 ,信号肽和成熟蛋白质的组成对蛋白质的转运都有影响 .TatA、TatB、TatC和TatE四种蛋白质参与了大肠杆菌的Tat转运系统 .被转运的底物蛋白质绝大多数为与细菌厌氧呼吸有关的含氧化还原辅因子的酶 ,并以折叠形式转运 .
文摘为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中,成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达,通过改变培养温度和IPTG浓度等条件,得到了能够大量表达目标蛋白的重组子。结果表明:大肠杆菌BL21(DE3)是gsiB基因表达的最佳宿主菌;18℃低温诱导培养有利于gsiB基因的大量表达;0.1mmol/LIPTG足够诱导gsiB基因表达,增加IPTG浓度(0.1mmol/L～1.0mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达,其分子量大小与预期相符。
文摘Background: CACNA1A encodes Cav2.1, the pore- forming subunit of P/Q- type voltage- gated calcium channel complexes. Mutations in CACNA1A cause a wide ran ge of neurologic disturbances variably associated with cerebellar degeneration. Functional studies to date focus on electrophysiologic defects that do not adequ ately explain the phenotypic findings. Objective: To investigate whether some mi ssense mutations might interfere with protein folding and trafficking, eventuall y leading to protein aggregation and neuronal injury. Methods: The authors studi ed the functional consequences of two pore missense mutations, C287Y and G293R, in two families with EA2, one newly discovered and the other previously reported . Both mutations caused episodic and interictal ataxia. The biophysical properti es of mutant and wild type calcium channels were examined by whole- cell patch - clamp recordings in transfected COS- 7 cells. The plasma membrane targeting was visualized by confocal fluorescence imaging on Cav2.1 tagged with green fluo rescent protein. Results: The mutant channels exhibited a marked reduction in cu rrent expression and deficiencies in plasma membrane targeting. Conclusions: In addition to altered channel function, the deficiency in protein misfolding and t rafficking associated with the C287Y and G293R mutants may contribute to the slo wly progressive cerebellar ataxia.
