Coexpression of DNA
Fragmentation Factor Subunits in E. coli by Two
Incompatible Plasmids
YANG Wei, ZHANG Lan, LU Zhi-Gang, ZHAI Zhong-He*
( College of Life Sciences, Peking University, Beijing 100871,
China )
Abstract 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.
Key words DFF45； DFF40； coexpression； incompatible plasmids； E.coli
*Corresponding author： Tel, 86-10-62754293； Fax, 86-10-62751850； e-mail, [email protected]