Journal Article

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

Brown, Nicholas G., VanderLinden, Ryan, Veld, Pim J. Huis in ’t, Lampert, Fabienne, Westermann, Stefan, Schulman, Brenda A., Petzold, Georg, Peters, Jan-Michael, Stark, Holger, Weissmann, Florian

2016

Overview

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.

• Publisher: National Academy of Sciences
• Subject: Baculoviridae - genetics / Baculovirus / Biochemistry / Biological Sciences / Cloning, Molecular - methods / Deoxyribonucleic acid / DNA / Gene expression / Genetic Vectors - genetics / Genomes / Multigene Family - genetics / Multiprotein Complexes - genetics / Multiprotein Complexes - metabolism / Mutagenesis, Site-Directed - methods / PNAS Plus / Protein Engineering - methods / Protein Subunits - genetics / Protein Subunits - metabolism / Proteins / Recombinant Proteins - metabolism / Transfection - methods / Viral Proteins - genetics / Viral Proteins - metabolism