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

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)622-630
Number of pages9
JournalMolecular Biotechnology
Volume61
Issue number8
DOIs
Publication statusPublished - Aug 1 2019

Fingerprint

Type I DNA Topoisomerase
Vaccinia virus
Viruses
Purification
DNA
DNA Topoisomerases
Adenosine Triphosphate
Superhelical DNA
Bombyx
Recombinant DNA
Baculoviridae
Transcription
Post Translational Protein Processing
Chromatography
Eukaryota
Adenosinetriphosphate
Escherichia coli
Nucleic Acids
Genetic Recombination
Larva

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

Cite this

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System. / Xu, Jian; Lee, Jae Man; Tatsuke, Tuneyuki; Ebihara, Takeru; Masuda, Akitsu; Hino, Masato; Morokuma, Daisuke; Fujita, Ryosuke; Mon, Hiroaki; Kusakabe, Takahiro; Takahashi, Masateru.

In: Molecular Biotechnology, Vol. 61, No. 8, 01.08.2019, p. 622-630.

Research output: Contribution to journalArticle

Xu, Jian ; Lee, Jae Man ; Tatsuke, Tuneyuki ; Ebihara, Takeru ; Masuda, Akitsu ; Hino, Masato ; Morokuma, Daisuke ; Fujita, Ryosuke ; Mon, Hiroaki ; Kusakabe, Takahiro ; Takahashi, Masateru. / Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System. In: Molecular Biotechnology. 2019 ; Vol. 61, No. 8. pp. 622-630.
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