红霉素合成蛋白基因的克隆和高表达

Cloning and High Expression of Erythromycin Synthetic Protein Gene

文献报道红霉素合成蛋白DEBS2(6-脱氧红霉内酯B合酶)(374 kD)基因在大肠杆菌(Escherichia coli)中异源表达量比较低,难以开展后续蛋白纯化和结构等研究。为获得该大蛋白基因的异源高表达,本研究通过PCR的方法,对编码红霉素合成蛋白DEBS2的基因起始5'端设计了起始密码子之后的十一个简并密码子序列,随机筛选获得在表达宿主BL21(DE3)中高表达的简并序列质粒,质粒命名为DEBS2-17。结果表明:mRNA的起始二级结构影响200 kD以上的蛋白质表达水平。本研究为开展红霉素合成蛋白的结构及生化研究提供理论基础,同时对表达分子量200 kD以上蛋白具有一定借鉴意义。

The literature reports that the erythromycin synthesis protein DEBS2(6-deoxyerythronolide B synthase)(374 kD)gene has a relatively low expression level in Escherichia coli,making it difficult to carry out subsequent purification and structure studies of the translated product.In order to obtain high expression of large proteins in heterologous host,this study designed eleven degenerate codons after the initial codon by PCR.The degenerate sequence began at the 5'end of the gene for erythromycin synthetic protein DEBS2.Degenerate plasmids with high expression in the expression host BL21(DE3)was obtained by random screening,and the plasmid was named DEBS2-17.The result showed that the initial secondary structure ofmRNAaffected protein expression level above 200 kD.This study provided a theoretical basis for the structural and biochemical research of erythromycin synthetic protein,and had a reference significance for the expression of proteins with molecular weight of more than 200 kD.