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.
Highlights
With the rapid development of single-particle cryo-electron microscopy, more and more macromolecular machineries structures, e.g. spliceosome (Nguyen et al, 2016; Yan et al, 2015a), ryanodine receptor, anaphase promoting complex (Chang et al, 2015), light harvest complex (Wei et al, 2016b) and mitochondrial respirasome (Gu et al, 2016; Letts et al, 2016), have been solved to near-atomic resolution, which have been waiting for many years
In order to overcome the difficulties in building large plasmids with conventional cloning methods, we incorporated the broadly applicable univector plasmid-fusion system (UPS) strategy (Liu et al, 1998) into our SmartBac vector system
Selection for the UPS recombination products is achieved by selecting for kanamycin resistance (KanR) after transformation into a pir- strain; the KanR gene in the donor vector can be expressed in a pir-background only when covalently linked to an acceptor that has a functional origin of replication (Liu et al, 1998)
Summary
With the rapid development of single-particle cryo-electron microscopy (cryo-EM), more and more macromolecular machineries structures, e.g. spliceosome (Nguyen et al, 2016; Yan et al, 2015a), ryanodine receptor (des Georges et al, 2016; Wei et al, 2016a; Yan et al, 2015b), anaphase promoting complex (Chang et al, 2015), light harvest complex (Wei et al, 2016b) and mitochondrial respirasome (Gu et al, 2016; Letts et al, 2016), have been solved to near-atomic resolution, which have been waiting for many years. In the first strategy insect cells are infected with multiple types of baculoviruses, each of which carries one or two gene expression cassettes (GECs) This strategy, which involves molecular cloning, is relatively simple and has been successfully applied by many research groups using the pFastBac series vectors The second strategy used to express multiprotein complexes is to construct a transfer plasmid carrying multiple GECs. The commercial pFastbac-Dual vector features two promoters for expression of two proteins simultaneously. In vivo processing of the polyprotein allows the proper assembly of the multiprotein complex This method is very good for balancing expression levels and achieving the correct subunit stoichiometry (Vijayachandran et al, 2011). We expect this system will aid structural and functional studies of large multiprotein complexes in the future
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