RT Journal Article
SR Electronic
T1 Large protein complex production using the SmartBac System - Strategies and Applications
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 219246
DO 10.1101/219246
A1 Zhai, Yujia
A1 Zhang, Danyang
A1 Yu, Leiye
A1 Sun, Fang
A1 Sun, Fei
YR 2017
UL http://biorxiv.org/content/early/2017/11/14/219246.abstract
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.