The light harvesting chlorophyll a/b-binding protein is one of key proteins in the transformation from light energy to chemical energy. An open reading frame coding precursor protein of cab gene was cloned from the fi...The light harvesting chlorophyll a/b-binding protein is one of key proteins in the transformation from light energy to chemical energy. An open reading frame coding precursor protein of cab gene was cloned from the first strand of bamboo cDNA through RT-PCR methods,and named as cab-PhE1 (cab gene 1 from Phyllostachys edulis EF207229). The sequence analysis showed that the deduced polypeptide was highly homologous to some other CAB proteins from monocotyledon,and the gene belonged to lhcb2 family. Tissue specific expression showed that cab-PhE1 expressed higher in leaf than sheath and stem. The prokaryotic expression vector of cab-PhE1 gene encoding the mature protein was constructed by subcloning the fragment into pET-23 a and was expressed in Escherichia coli induced by IPTG. The molecular weight of the induced protein was about 28 ku,approximate to that of the mature protein. This work is a key to the further research on in vitro reconstitution of light-harvesting Chl a/b complexes.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
文摘The light harvesting chlorophyll a/b-binding protein is one of key proteins in the transformation from light energy to chemical energy. An open reading frame coding precursor protein of cab gene was cloned from the first strand of bamboo cDNA through RT-PCR methods,and named as cab-PhE1 (cab gene 1 from Phyllostachys edulis EF207229). The sequence analysis showed that the deduced polypeptide was highly homologous to some other CAB proteins from monocotyledon,and the gene belonged to lhcb2 family. Tissue specific expression showed that cab-PhE1 expressed higher in leaf than sheath and stem. The prokaryotic expression vector of cab-PhE1 gene encoding the mature protein was constructed by subcloning the fragment into pET-23 a and was expressed in Escherichia coli induced by IPTG. The molecular weight of the induced protein was about 28 ku,approximate to that of the mature protein. This work is a key to the further research on in vitro reconstitution of light-harvesting Chl a/b complexes.
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。
