利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40  

Coexpression of DNA Fragmentation Factor Subunits in E.coli by Two Incompatible Plasmids

作　　者：杨巍[1] 张岚[1] 卢智刚[1] 翟中和[1] 

机构地区：[1]北京大学生命科学学院,北京100871

出　　处：《生物化学与生物物理学报》2001年第2期238-242,共5页

基　　金：国家重点基础研究发展规划项目!No.G19990 5 390 5&&

摘　　要：DNA断裂因子 (DNAfragmentationfactor,DFF)是细胞凋亡过程中起重要作用的蛋白质之一 ,它由分子量为 45kD和 40kD的两个亚基构成 ,分别称为DFF45和DFF40。利用RT PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA ,分别克隆到卡那霉素抗性表达载体pET 2 8a(+ )中 ,构建了pET2 8a DFF45和 pET2 8a DFF40。用它们单独转化大肠杆菌BL2 1(DE3)后 ,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的 5 6 %和 2 2 %。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET 2 1a(+ )中 ,得到了pET2 1a DFF45。利用二者的不同抗性 ,将pET2 8a DFF40和 pET2 1a DFF45共同转化大肠杆菌BL2 1(DE3) ,工程菌经IPTG诱导后实现了DFF45和DFF40的共表达 ,表达产物各约占菌体总蛋白质的30 %和 17%。为了研究这两种不相容质粒在细菌中共存的稳定性 ,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养 14h ,发现此时仍有 75 %以上的细菌可同时耐受两种抗生素 ,即同时含有 pET2 8a DFF45和 pET2 8a DFF40 。The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD (DFF40/CAD) and 45 kD(DFF45/ICAD) subunits. Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase activated DFF40 nuclease, which is inhibited by a chaperone like subunit DFF45. In this work, the coding regions of human DFF 45 and DFF 40 mRNAs were amplified from total RNA of HeLa cells by RT PCR. Two 1 kb DNA fragments were obtained and were cloned into a kanamycin resistant bacterial expression vector, pET 28a(+), generating pET28a DFF 45 and pET28a DFF 40, which were then used to transform E.coli BL21(DE3), respectively. After induction with IPTG, DFF45 and DFF40 were expressed effectively, accounting for about 56% and 22% of total bacterial proteins, respectively. Since successful expression of properly folded DFF40 requires coexpression with DFF45, the full length DFF 45 cDNA was inserted into an ampicillin resistant expression vector, pET 21a(+), and the recombinant plasmid was designated pET21a DFF 45. Under screening pressure by ampicillin and kanamycin simultaneously, E.coli BL21(DE3) was cotransformed with pET21a DFF 45 and pET28a DFF 40. Upon induction with IPTG, DFF45 and DFF40 were coexpressed efficiently and the desired products comprised about 30% and 17% of total cell proteins, respectively. To further study the stability of the two incompatible plasmids' coexistance in E.coli , the cotransformant was cultured in liquid medium containing ampicillin and kanamycin for 14 h, and more than 75% of the cells were found to be resistant to the two antibiotics, that is, they carried both pET21a DFF 45 and pET28a DFF 40. Thus, a novel method of coexpressing different proteins using two incompatible plasmids was developed.

关 键 词：DNA断裂因子(DFF)45 DNA断裂因子(DFF)40 共表达 不相容质粒 大肠杆菌 

分 类 号：Q786[生物学—分子生物学]