Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts.
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 6xhistidine 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 lambdaDNA 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.
Yang
Wei
W
College of Life Sciences, Peking University, Beijing 100871, China. zhaizh@plum.lsc.pku.edu.cn
Lu
Zhi-Gang
ZG
Zhang
Lan
L
Zhai
Zhong-He
ZH