Gene Cloning And Expression, Enzymatic Purification And Characterization Identification Of Chlamydia Pneumoniae RNase H Ⅱ

Chlamydia species are obligate intracellular pathogens, which cause a broad range of diseases in humen and animals, having not genetic operation system. It is a reasonable method of researthing Chlamydia biology by in vitro identifying biological functions of Chlamydia proteins which were expressed in biology, such as Escherichia coli etc. RNase H enzymes are ribonucleotide-specific endonucleases that cleave the RNA portion of RNA-DNA/DNA or RNA/DNA duplex, which activities depend on a divalent positive ion, releasing oligoribonucleotides with 3′-OH and 5′-Phosphate ends. RNase H enzymes have been identified in all three kingdoms of life such as virus, prokaryote and eukaryote. Single bacterial and eukaryotic cells often contain two different RNases H, which show little sequence similarity with each other. Based on the difference in the amino acid sequences, RNases H are classified into two major families, Type 1 and Type 2 RNases H. Analysing the whole genome sequence of Chlamydia pneumoniae AR39, there does not exist genes encoding RNase HI, but having two genes encoding RNase HII with different sequences:CP0654 and CP0782. The enzymes which CP0645 and CP0782 encode were named as CpRNase HIIa and CpRNase HIIb respectively. CP0645 and CP0782 gene were obtained by PCR using chlamydial genomic DNA as template. Adopting normal DNA recombination protocols: digestion with restriction endonucleases and ligation with T4-DNA ligase, the two genes were cloned into expression plamid pET-28a of Escherichia coli. DNA sequencing confirmed that the constructs did have Chlamydial encoding genes in it which were correct in translation frame and sequence. Consulting the application manual of Novagen company, recombinant CpRNase HIIa and CpRNase HIIb were expressed and purified. The soluble proteins were 20 % of total expressed CpRNase HIIa and 65 % of total expressed CpRNase HIIb in Escherichia coli BL21(DE3) cells, respectively. Native purification and denaturing Ni-NTA affinity chromatography purifications were performed to recover the recombinant CpRNase HIIs from induced bacteria. Proteins gained by native Ni-NTA purification were active, while proteins recovered by denaturing Ni-NTA purification were inactive which would be active by re-naturation. Simple dialysis with gradiently-decreased concentrations of urea could generate CpRNase HIIa and CpRNase HIIb as active as those recovered by native Ni-NTA purification. The optimal pH, dependence on metal ion and specific enzymatic cleavage sites of CpRNase HIIa and CpRNase HIIb were confirmed furthermore. The optimum pH of CpRNase HIIa and CpRNase HIIb were pH 9-10. The enzymatic activity of CpRNase HIIa and CpRNase HIIb depended on divalent metal ion, which was similar with identified RNases HII and with a preference for Mg2+ and Mn²⁺. CpRNase HIIa cleaved 12 bp RNA/DNA substrate at multiple sites while CpRNase HIIb only one cleavage site. CpRNase HIIb could not cleavage 35 bp chimeric DNA-RNA-DNA/DNA substrates while CpRNase HIIa could cleavage it, but match or mismatch of base pair in hybrid double strand had obvious effections on its hydrolysis efficiency. In general, we successfully finished the following work such as: 1 Two RNase H genes were cloned from chlamydial genome. 2 Proteins encoded by the two genes were expressed. 3 CpRNase HIIs with catalysis functions were purified. 4 Enzymatic characterizations of the two proteins were identified also.