Recombinant Human DFF45
Inhibits Apoptosis specific Endonuclease in a Cell-free System of Xenopus Egg Extracts
YANG Wei, LU Zhi-Gang, ZHANG Lan, 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 and 45 kD subunits.
The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the
genomic DNA degradation into nucleosomal fragments during apoptosis. As an inhibitor
for DFF40, the 45 kD subunit (DFF45) complexes with DFF40,inhibiting DNase
activity until certain apoptosis signals are received. In cells undergoing
apoptosis, the cleavage of DFF45 by activated caspase-3 frees DFF40from the
complex and initiates the apoptosis-specific DNA fragmentation. In this report,
the coding region of human DFF45 gene was amplified from the total RNA
of HeLa cells by RT-PCR. The resulting 1 kb DNA fragment was cloned into the
bacterial expression vector pET-28a(+) with a 6×histidine tag fused to the
N-terminus of DFF45, generating plasmid pET28a-DFF45, which was then
used to transform E.coli BL21(DE3). Induced by IPTG, the recombinant
DFF45 was expressed efficiently with a yield of 56.6% of total bacterial
proteins. The product was purified to homogeneity through a nickel affinity
column, followed by heat treatment, and approximately 4–6 mg of DFF was
purified from 100 ml culture. Purified recombinant human DFF45, added into the
apoptotic cell-free system of Xenopus egg extracts, could effectively
inhibit both the digestion of λDNA and the degradation of chromosomal DNA into
nucleosomal fragments in the nuclei of chicken red blood cells. Our results
demonstrated the existence of an apoptosis specific endonuclease in this cell-free
system, the activity of which could be inhibited by recombinant human DFF45.
Key words apoptosis；DFF45；cell-free system；cytochrome c；apoptosis-specific
endonuclease
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