Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system

• Published in: Applied microbiology and biotechnology 2013-08, Vol.97 (15), p.6813-6822
• Main Authors: Mon, Hiroaki, Lee, JaeMan, Fukushima, Mai, Nagata, Yudai, Fujii, Mie, Xu, Jian, Nishi, Oumi, Iiyama, Kazuhiro, Kusakabe, Takahiro
• Format: Article
• Language: English
• Subjects: Animals; Apium graveolens; Apium graveolens - enzymology; Baculoviridae - genetics; Baculovirus; Base Sequence; Biological research; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Bombyx - genetics; Bombyx mori; DNA Primers; DNA Restriction Enzymes - metabolism; Enzymes; Glycerol; Glycosylation; Life Sciences; Microbial Genetics and Genomics; Microbiology; Mutagenesis; Mutation; Nucleases; Plasmids; Polymorphism; Proteins; Silk; Studies; Yeast
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-4583-1

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.