Cloning, expression, and characterization of uracil-DNA glycosylase of Chlamydia pneumoniae in Escherichia coli.
A uracil-DNA glycosylase gene was cloned from Chlamydia pneumoniae AR39 and expressed in E. coli strains BL21 (DE3) and BL21 (DE3) pLysS. After purification by Ni-NTA His x Bind Resin and DEAE Sepharose Fast Flow column chromatography, recombinant CpUDG with a specific activity of 1,000,000 U/mg was obtained. The enzymatic activity of the purified CpUDG protein was further characterized using oligodeoxyribonucleotides carrying uracil bases as substrates. The base opposite to uracil in double strand DNAs affected uracil removal efficiencies in the order: U/- > U/T > U/C > U/G > U/A. Free uracil and abasic sites (AP site) could inhibit the reaction. The optimal temperature and pH for uracil removal by CpUDG were 37 degrees C and pH 8.0, respectively. Site-directed mutagenesis studies indicated that amino acids D77, H200, and A205 were important for the catalytic activity of CpUDG. Together, these data suggest that CpUDG is a member of the UDG family-I protein. This is the first report on cloning, expression, and characterization of Chlamydia uracil-DNA glycosylase.
Liu
Xipeng
X
College of Life Sciences and Technology, Shanghai Jiaotong University, No. 1954 Hua-Shan Road, Shanghai 200030, China. liu-xipeng@163.com
Liu
Jianhua
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016993:Chlamydophila pneumoniae / Q000201:enzymology*; D003001:Cloning, Molecular; D045647:DNA Glycosylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase