Applications of Golden Gate cloning to protein production using the baculovirus expression vector system

Advances in structural biology techniques over the last decades have made it increasingly possible to determine the structures of multi-protein complexes. Generation of sufficient recombinant material for such studies remains a bottleneck and often requires screening a variety of purification strategies and different subunit compositions to reproducibly isolate homogeneous complexes. Parallel advances in molecular biology now make it possible to easily generate panels of constructs with different affinity tags and different multi-protein components. Here, we describe two protocols based on Golden Gate cloning, which facilitate the generation of multi-protein complexes for protein production via the Baculovirus Expression Vector System. This robust method makes it possible to efficiently generate a panel of multi-gene expression constructs containing up to 15 open reading frames.