Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System

Jian Xu, Jae Man Lee, Tuneyuki Tatsuke, Takeru Ebihara, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Masateru Takahashi

研究成果: Contribution to journalArticle査読

2 被引用数 (Scopus)

抄録

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.

本文言語英語
ページ(範囲)622-630
ページ数9
ジャーナルMolecular Biotechnology
61
8
DOI
出版ステータス出版済み - 8 1 2019

All Science Journal Classification (ASJC) codes

  • バイオテクノロジー
  • バイオエンジニアリング
  • 生化学
  • 応用微生物学とバイオテクノロジー
  • 分子生物学

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