TY - JOUR
T1 - Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system
AU - Mon, Hiroaki
AU - Lee, Jaeman
AU - Fukushima, Mai
AU - Nagata, Yudai
AU - Fujii, Mie
AU - Xu, Jian
AU - Nishi, Oumi
AU - Iiyama, Kazuhiro
AU - Kusakabe, Takahiro
N1 - Funding Information:
Acknowledgments We thank Dr. Susumu Shimizu for the gift of reagents. This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Project for Plant, Insect and Animal using Genome Technology INSECT-1201), and KAKENHI nos. 22248003, 22248004, and 23580077 from the Japan Society for the Promotion of Science.
PY - 2013/8
Y1 - 2013/8
N2 - 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 Ca2+ and Sr2+ were able to replace Mg2+ 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.
AB - 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 Ca2+ and Sr2+ were able to replace Mg2+ 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.
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U2 - 10.1007/s00253-012-4583-1
DO - 10.1007/s00253-012-4583-1
M3 - Article
C2 - 23179626
AN - SCOPUS:84880509955
SN - 0175-7598
VL - 97
SP - 6813
EP - 6822
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 15
ER -