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SmartBac, a new baculovirus system for large protein complex production

Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and...

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Detalles Bibliográficos
Autores principales: Zhai, Yujia, Zhang, Danyang, Yu, Leiye, Sun, Fang, Sun, Fei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173262/
https://www.ncbi.nlm.nih.gov/pubmed/32337507
http://dx.doi.org/10.1016/j.yjsbx.2019.100003
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author Zhai, Yujia
Zhang, Danyang
Yu, Leiye
Sun, Fang
Sun, Fei
author_facet Zhai, Yujia
Zhang, Danyang
Yu, Leiye
Sun, Fang
Sun, Fei
author_sort Zhai, Yujia
collection PubMed
description Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmid. The fluorescent proteins are designed co-expressed with the target to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for efficient purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed and purified, suggesting a great potential of SmartBac system for its wide application in the future.
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spelling pubmed-71732622020-04-22 SmartBac, a new baculovirus system for large protein complex production Zhai, Yujia Zhang, Danyang Yu, Leiye Sun, Fang Sun, Fei J Struct Biol X Article Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmid. The fluorescent proteins are designed co-expressed with the target to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for efficient purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed and purified, suggesting a great potential of SmartBac system for its wide application in the future. Elsevier 2019-02-10 /pmc/articles/PMC7173262/ /pubmed/32337507 http://dx.doi.org/10.1016/j.yjsbx.2019.100003 Text en © 2019 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhai, Yujia
Zhang, Danyang
Yu, Leiye
Sun, Fang
Sun, Fei
SmartBac, a new baculovirus system for large protein complex production
title SmartBac, a new baculovirus system for large protein complex production
title_full SmartBac, a new baculovirus system for large protein complex production
title_fullStr SmartBac, a new baculovirus system for large protein complex production
title_full_unstemmed SmartBac, a new baculovirus system for large protein complex production
title_short SmartBac, a new baculovirus system for large protein complex production
title_sort smartbac, a new baculovirus system for large protein complex production
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173262/
https://www.ncbi.nlm.nih.gov/pubmed/32337507
http://dx.doi.org/10.1016/j.yjsbx.2019.100003
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