Expression of both Chlamydia pneumoniae RNase HIIs in Escherichia coli.
Both genes encoding the RNase HIIs from Chlamydia pneumoniae AR 39 (discriminated as CpRNase HIIa and CpRNase HIIb in this report) were cloned and efficiently expressed in Escherichia coli. These genes amplified from Chlamydial genomes with PCR were digested with restriction endonucleases and then cloned into plasmid pET-28a predigested with the same enzymes. DNA sequencing confirmed that the constructs were correct in translation frame and coding sequence. Recombinant RNase HIIs were over-expressed by 0.5 mM IPTG induction. CpRNase HIIa existed mainly as inclusion bodies while CpRNase HIIb mainly as soluble fractions in E. coli. The soluble proteins were 20% of total expressed CpRNase HIIa and 65% of total expressed CpRNase HIIb, respectively. Native purification and denaturing Ni-NTA purification were performed to recover the recombinant CpRNase HIIs from induced bacteria. 3.36 mg CpRNase HIIa and 18 mg CpRNase HIIb were, respectively, obtained from 1 g wet bacteria with native Ni-NTA purification. Denaturing Ni-NTA purification recovered 14.48 mg CpRNase HIIa and 10.4 mg CpRNase HIIb from 1 g wet bacteria, respectively. Although the proteins recovered by denaturing Ni-NTA purification were inactive, re-folding by dialysis against decreased concentrations of urea could generate CpRNase HIIa and CpRNase HIIb as active as those recovered by native Ni-NTA purification. These efforts offered basis for further study on the structure-function relationships and their biological importance of Chlamydial RNase HIIs.
Pei
Dongli
D
College of Life Sciences and Technology, Shanghai Jiaotong University, No. 1954 Hua-Shan Road, Shanghai 200030, China.
Liu
Jianhua
J
Liu
Xipeng
X
Li
Suoping
S
D001483:Base Sequence; D002413:Cations, Divalent; D016993:Chlamydophila pneumoniae / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D011994:Recombinant Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H