MultiBac: expanding the research toolbox for multiprotein complexes

Abstract

Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…The ACEMBL technology was conceptualized to meet these requirements, to enable structural and functional "complexomics" research and discovery [31][32][33][34]. ACEMBL comprises recombination-based assembly of DNA elements into functional multigene expression constructs that can be rapidly permutated in a combinatorial fashion [33].…”

“…Subsequently, efficient ACEMBL tool-kits have been developed also for multigene expression in eukaryotic hosts [34][35][36][37]. The integration of the ACEMBL technology in MultiBac, currently the lead technology for multiprotein complex production in insect cells, is described in a dedicated contribution of this issue [37].…”

“…Recombinant production offers solutions to these impediments, and a wide range of expression systems are available to produce proteins recombinantly in prokaryotic and eukaryotic hosts [31,33,[34][35][36][37][38][39][40][41][42][43][44][45]. Recombinant expression systems share in common that one or several DNA segments encoding for proteins, protein domains or multicomponent protein complexes are typically combined with DNA elements including DNAs that control transcription (promoters, terminators, others) and translation (ribosome binding sites, ShineDalgarno sequences, Kozak consensus sequences, enhancers, others) and inserted into a functional DNA module (plasmid, cosmid, artificial chromosome, genome, others).…”

“…The ACEMBL system utilizes a series of custom-designed vectors (called Acceptor or Donor, respectively) for multigene vector generation catalyzed by Cre-LoxP recombination [33,34,44,46,47]. All ACEMBL vectors are scratch-built, synthetic small plasmids (2-3 kilobases).…”

“…Multifusion plasmids generated from Cre-LoxP reactions are utilized by the ACEMBL-derived MultiMam system to facilitate simultaneous multigene introduction into mammalian cells [35,36] (see also 3.2). The MultiBac baculovirus / insect cell system has been upgraded for robotics by incorporating ACEMBL DNA modules (MIE and HE / BstXI sites) for automatable and theoretically unlimited multigene insertion into a baculoviral genome for protein co-expression in insect cells [34] (see also Chapter on MultiBac by Sari and co-workers in this issue). Selected examples of ACEMBL applications are highlighted in the following.…”