A platform for research: civil engineering, architecture and urbanism
A platform for research: civil engineering, architecture and urbanism
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 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. Keywords: Baculovirus system, Cre recombination, Gibson assembly, Polyprotein strategy, Protein complex production
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 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. Keywords: Baculovirus system, Cre recombination, Gibson assembly, Polyprotein strategy, Protein complex production
SmartBac, a new baculovirus system for large protein complex production
Yujia Zhai (author) / Danyang Zhang (author) / Leiye Yu (author) / Fang Sun (author) / Fei Sun (author)
2019
Article (Journal)
Electronic Resource
Unknown
Metadata by DOAJ is licensed under CC BY-SA 1.0
Baculovirus Pesticides: More Uncertainties about Field Trials
| Online Contents | 1995
Tandem purification of Mre11/Rad50-dimers using the baculovirus expression system
| UB Braunschweig | 2012