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KMID : 0311119990400010061
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Yonsei Medical Journal
1999 Volume.40 No. 1 p.61 ~ p.68
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Prokaryotic Expression and Characterization of Human AP DNA Endonuclease
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Sang Hwan Oh
Dong Weon Song/Mi Young Lee
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Abstract
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The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 μM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.
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KEYWORD
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AP DNA endonuclease, expression, E. coli, characterization, DNA repair,
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