PMID:10788800
| Citation |
Ohtani, N, Haruki, M, Muroya, A, Morikawa, M and Kanaya, S (2000) Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form. J. Biochem. 127:895-9 |
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| Abstract |
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. |
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| Keywords |
Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; DNA, Viral/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Nucleic Acid Heteroduplexes/metabolism; RNA, Viral/metabolism; Recombinant Proteins/biosynthesis; Ribonuclease H/biosynthesis; Ribonuclease H/genetics; Ribonuclease H/metabolism; Sequence Analysis, Protein; Solubility |
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