title: Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system.
abstract: Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca(2+) and Sr(2+) were able to replace Mg(2+) and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.
authors.affiliation: Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mon
authors.forename: Jaeman
authors.initials: J
authors.lastname: Lee
authors.forename: Mai
authors.initials: M
authors.lastname: Fukushima
authors.forename: Yudai
authors.initials: Y
authors.lastname: Nagata
authors.forename: Mie
authors.initials: M
authors.lastname: Fujii
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Oumi
authors.initials: O
authors.lastname: Nishi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iiyama
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kusakabe
mesh_terms: D000818:Animals; D000077102:Apium / Q000201:enzymology*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D006031:Glycosylation
chemical_list: D017931:DNA Primers; D004262:DNA Restriction Enzymes