Research

Schemes for the expression of large multiprotein complexes. (a) The eight-subunit protein complex to be expressed. The eight genes are divided into two groups according to their sizes. Two long polyproteins are designed with TEV cleavage sites separating the adjacent genes. Represents (b) Schematic representation of Scheme 1 for the expression of multiprotein complexes with a molecular weight less than 600？kDa. Here the acceptor vector 4V1R is used, but 5V1TR can also be used.
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.
Reference:
Zhai Y.*, Zhang D., Yu L., Sun Fang and Sun F.* (2019), SmartBac, a new baculovirus system for large protein complex production. Journal of Structural Biology:X, 100003. doi: 10.1016/j.yjsbx.2019.100003.