Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System
Lee, Jae Man
Online Publication Date2019-06-05
Print Publication Date2019-08
Embargo End Date2020-06-05
Permanent link to this recordhttp://hdl.handle.net/10754/656411
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AbstractType 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.
CitationXu, J., Lee, J. M., Tatsuke, T., Ebihara, T., Masuda, A., Hino, M., … Takahashi, M. (2019). Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System. Molecular Biotechnology, 61(8), 622–630. doi:10.1007/s12033-019-00184-4
SponsorsWe thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for kindly providing the NIAS-Bm-oyanagi2 (BmO2) cell line. This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program).