Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form.
Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.
Ohtani
N
N
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
Haruki
M
M
Muroya
A
A
Morikawa
M
M
Kanaya
S
S
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D016914:Ribonuclease H / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012995:Solubility
D001426:Bacterial Proteins; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral; D011994:Recombinant Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H; C078856:ribonuclease HI