层理鞭枝藻藻红蓝蛋白操纵子F基因的克隆和表达

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CLONING AND OVEREXPRESSING OF PHYCOERYTHROCYANIN OPERON F-GENE OF MASTIGOCLADUS LAMINOSUS IN ESCHERICHIA COLI

摘要: 以层理鞭枝藻总DNA为模板,用PCR技术扩增得到藻红蓝蛋白操纵子F基因(pecF),然后克隆于质粒pBluescript sk(+).将pecF基因亚克隆于表达载体pGEMD,所形成重组质粒pGEMD-pecF在E.coli BL21(DE3)中获得10%外源表达蛋白并形成包涵体.这个表达产物的分子量为22.5kDa,与按DNA序列预测的蛋白分子量一致.经藻红蓝蛋白α-亚基的重组实验证明,该pecF基因编码的表达产物PecF与藻红蓝蛋白操纵子E基因(pecE)的表达产物PecE共同存在时,具有藻红蓝蛋白裂合酶活性.

Abstract: By PCR method, apo phycoerythrocyanin operon F gene(pecF)of Mastigocladus laninosus was amplified from its genomic DNA,and then cloned in pBluescript The pecF gene was subcloned into the expression vector pGEMD,and then transformed into E.coli BL21(DE3)After induction, a new protein of molecular weight 22 5kDa existing in inclusion body was overexpressed The expression product was confirmed to possess phycoerythrocyanin lyase activity