PT  - JOURNAL ARTICLE
AU  - Zhai, Yujia
AU  - Zhang, Danyang
AU  - Yu, Leiye
AU  - Sun, Fang
AU  - Sun, Fei
TI  - Large protein complex production using the SmartBac System - Strategies and Applications
AID  - 10.1101/219246
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 219246
4099  - http://biorxiv.org/content/early/2017/11/14/219246.short
4100  - http://biorxiv.org/content/early/2017/11/14/219246.full
AB  - 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 plasmids. The fluorescent proteins are designed to be co-expressed with recombinant proteins to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for effective purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed, suggesting a great potential of SmartBac for its wide application in the future structural biology study.