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6568102 
Journal Article 
biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes 
Weissmann, F; Petzold, G; Vanderlinden, R; Huis In 't Veld, PJ; Brown, NG; Lampert, F; Westermann, S; Stark, H; Schulman, BA; Peters, JM 
2016 
1 
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
EISSN: 1091-6490 
113 
19 
E2564-E2569 
English 
27114506 
WOS:000375478800006 
Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.