Coexpression of DNA Fragmentation Factor Subunits in E.coli by Two Incompatible Plasmids.
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 DFF45 and DFF40 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-DFF45 and pET28a-DFF40, 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 DFF45 cDNA was inserted into an ampicillin resistant expression vector, pET-21a( ), and the recombinant plasmid was designated pET21a-DFF45. Under screening pressure by ampicillin and kanamycin simultaneously, E.coli BL21(DE3) was cotransformed with pET21a-DFF45 and pET28a-DFF40. 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-DFF45 and pET28a-DFF40. Thus, a novel method of coexpressing different proteins using two incompatible plasmids was developed.
Yang
Wei
W
College of Life Sciences, Peking University, Beijing 100871, China. zhaizh@plum.lsc.pku.edu.cn
Zhang
Lan
L
Lu
Zhi-Gang
ZG
Zhai
Zhong-He
ZH