SmartBac, a new baculovirus system for large protein complex production.
J Struct Biol X
; 1: 100003, 2019.
Article
em En
| MEDLINE
| ID: mdl-32337507
ABSTRACT
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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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
J Struct Biol X
Ano de publicação:
2019
Tipo de documento:
Article