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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2019
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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. |
format | Online Article Text |
id | pubmed-7173262 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
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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