MultiBac: Multigene Baculovirus‐Based Eukaryotic Protein Complex Production

Multiprotein complexes are an emerging focus in current biology, resulting in a demand for advanced heterologous expression systems. This unit provides protocols for the expression of eukaryotic multiprotein complexes using multigene expression vectors. Homologous and site-specific recombinases facilitate their assembly. Thus, modification of individual subunits for revised expression studies is achieved with comparative ease. The strategy outlined here employs the MultiBac baculoviral expression system for multiprotein complexes as an example. Baculoviral expression does not require particular safety precautions due to the replication incompetence of baculovirus in mammalian hosts. The MultiBac system provides for improved protein production due to deletion of specific viral genes (V-cath, chiA). Most of the steps described in this unit are tailored for high-throughput approaches. The general strategy of rapidly combining encoding DNAs by recombination into multigene expression vectors for protein complex expression can also be applied to other prokaryotic or mammalian expression systems.