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.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Ohtani N,Haruki M,Muroya A,Morikawa M,Kanaya Sdoi
10.1093/oxfordjournals.jbchem.a022684keywords:
subject
Has Abstractpub_date
2000-05-01 00:00:00pages
895-9issue
5eissn
0021-924Xissn
1756-2651journal_volume
127pub_type
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