Authors
Yan Nie, Christoph Bieniossek, Daniel Frey, Natacha Olieric, Christiane Schaffitzel, Michel O. Steinmetz, and Imre Berger
Summary
Multiprotein complexes are an emerging focus of contemporary biological research efforts1. Molecular and structural studies of multiprotein assemblies are often handicapped by the low abundance and heterogeneous nature of most of these complexes in their native hosts, thus inhibiting direct extraction. Recombinant methods that can achieve overproduction of these multiprotein complexes are therefore often a crucial prerequisite for their study. We addressed several of the challenges by creating ACEMBL, a system for rapid and flexible multigene assembly for multiprotein expression in E. coli. ACEMBL complements MultiBac, our previously introduced expression technology for the baculovirus/insect cell system2. ACEMBL uses recombineering for constructing multigene expression vectors and to rapidly introduce diversity into each gene of interest if the need arises. These features are especially important in modern structural biology, as rapid revision of complex expression and diversification of each component involved can be crucial for successful structure determination. The ACEMBL system can be fully automated, which is a top priority in current protein science. For further information about ACEMBL, including updates of the procedures used, a User Manual can be obtained from our EMBL home page: http://www.embl.fr/research/services/berger/ACEMBL.pdf. For ACEMBL reagents please contact [email protected]. The protocols presented in the following describe in detail the approaches for (de)constructing multigene expression vectors in the ACEMBL system: (1) Single gene insertion or polycistron assembly via sequence and ligation independent cloning (SLIC) procedures; (2) gene insertion by restriction/ligation; (3) expression cassette multiplication by using homing endonucleases (HE) and (4) fusion of multiple expression plasmids into a single multigene expression construct by site-specific recombination using the Cre recombinase. In addition to multigene construction, we also describe how to deconstruct multigene expression fusion plasmids by using the Cre enzyme, for example to change or alter only a particular subunit of a multiprotein complex. Combination of the protocols presented allows for simple assembly and disassembly of multigene constructs for multiprotein complex expression, as well as for rapid revision and diversification of expression experiments (Fig. 1). The protocols can be used in a manual setup and also in a robotic environment using a liquid handling workstation.Further details
The protocol was published on Protocol Exchange on 8 May 2009. To see the entire protocol, click on the source link.Advertisement
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