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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

The MultiBac Protein Complex Production Platform at the EMBL

Published: July 11th, 2013

DOI:

10.3791/50159

1EMBL Grenoble Outstation and Unit of Virus Host Cell Interactions (UVHCI) UMR5322

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Biological activity is controlled by assemblies of proteins and other biomolecules that act in concert to catalyze cellular functions. Notable examples include the machinery that transcribes the hereditary information contained in DNA into messenger RNA. In humans, more than 100 proteins come together in a defined and regulated process to transcribe genes, forming large multiprotein complexes with 10 and more subunits including RNA polymerase II and the general transcription factors such as TFIID, TFIIH and others.9 Other examples are the ribosome, consisting of many proteins and RNA molecules, that catalyzes protein synthesis, or the nuclear pore ....

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1. Tandem Recombineering (TR) for Creating Multigene Expression Constructs

  1. Planning the co-expression strategy. Design approach for inserting your genes of interest into Donors and Acceptors. Potential physiological submodules of your complex should be grouped together on specific Acceptors and Donors. Use Multiplication Module consisting of Homing endonuclease (HE) - BstXI pairs to combine expression cassettes on individual Donor and Acceptor plasmids.7,8 Create all rele.......

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Strong co-expression of heterologous proteins achieved by the MultiBac system is shown in Figure 1d (probes taken 48 hr after infecting a suspension cell culture). The overexpressed protein bands are clearly discernible in the whole cell extract (SNP) and the cleared lysate (SN). The quality and quantity of the protein material produced is often sufficient to enable structure determination of protein complexes, such as the mitotic checkpoint complex MCC shown in Figure 1e.17

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Video snap-shots in Figures 2 and 3 illustrate the entire process from robot-assisted generation from cDNA of multigene expression constructs all the way to infection of insect cell cultures for protein production. New reagents (plasmids and virus) and robust protocols have been developed to enable a pipeline relying on SOPs. The entire pipeline has been implemented as a platform technology at the EMBL in Grenoble. The MultiBac platform has been accessed by many scientists from academia .......

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We thank Christoph Bieniossek, Simon Trowitzsch, Daniel Fitzgerald, Yuichiro Takagi, Christiane Schaffitzel, Yvonne Hunziker, Timothy Richmond and all past and present members of the Berger laboratory for help and advice. The MultiBac platform and its development have been and are generously supported by funding agencies including the Swiss National Science Foundation (SNSF), the Agence National de Recherche (ANR) and the Centre National de Recherche Scientifique (CNRS) and the European Commission (EC) in Framework programs (FP) 6 and 7. Support for transnational access is provided by the EC FP7 projects P-CUBE (w....

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
Bluo-Gal Invitrogen 15519-028 (1 g)
Tetracycline Euromedex UT2965-B (25 g) 1,000X at 10 mg/ml
Kanamycine Euromedex EU0420 (25 g) 1,000X at 50 mg/ml
Gentamycine SIGMA G3632 (5 g) 1,000X at 10 mg/ml
IPTG Euromedex EU0008-B (5 g) 1,000X at 1M
Cre-recombinase New England BioLabs M0298
X-Treme GENE HP transfection reagent Roche 06 366 236 001
Hyclone SFM4 Insect Thermo Scientific SH 30913.02
6-well plate Falcon Dominique Dutscher 353046
2 ml pipette Falcon Dominique Dutscher 357507
5 ml pipette Falcon Dominique Dutscher 357543
10 ml pipette Falcon Dominique Dutscher 357551
25 ml pipette Falcon Dominique Dutscher 357535
50 ml pipette Falcon Dominique Dutscher 357550
50 ml tube Falcon Dominique Dutscher 352070
15 ml tube Falcon Dominique Dutscher 352096
1.8 ml cryotube Nunc Dominique Dutscher 55005
100 ml shaker flasks Pyrex Dominique Dutscher 211917
250 ml shaker flasks Pyrex Dominique Dutscher 211918
500 ml shaker flasks Pyrex Dominique Dutscher 211919
2 L shaker flasks Pyrex Dominique Dutscher 211921
Certomat Orbital Shaker + plateau Sartorius 4445110, 4445233
Liquid nitrogen tank dewar 35 L Fisher Scientific M76801
Biological Safety Cabinet Faster Sodipro FASV20000606
Optical Microscope Zeiss 451207
Sf21 Insect cells
Hi5 Insect cells Invitrogen B855-02
Tecan freedom EVO running Evoware plus TECAN
10 μl conductive tips (black), TECAN 10 612 516
200 μl conductive tips (black) TECAN 10 612 510
disposable trough for reagents, 100 ml TECAN 10 613 049
twin.tec PCR plate 96, skirted Eppendorf 0030 128.648
96 well V bottom, non sterile BD falcon 353263
96 deepwell plate color natural, PP) Fisher M3752M
PS microplate, 96 well flat bottom Greiner 655101
96 deepwell plate Thermo scientific AB-0932
24 well blocks RB Qiagen 19583
DpnI restriction enzyme NEB R0176L 20 U/uL
NEBuffer 4 10X NEB B7004S
2X phusion mastermix HF Finnzyme ref F-531L
2X phusion mastermix GC Finnzyme ref F-532L
DGLB 1.5X homemade 7.5% glycerol, 0.031% Bromophenol blue, 0.031% Xylen cyanol FF
High DNA Mass Ladder for e-gel Life Technologies 10496-016
Low DNA Mass Ladder for e-gel Life Technologies 10068-013
E-gel 48 1% agarose GP Life Technologies G8008-01
Nucleo Spin- robot-96 plasmid kit Macherey Nagel 740 708.24
PCR clean-up kit, Nucleospin Robot-96 Extract Macherey Nagel 740 707.2
Gotaq green master mix Promega M7113
T4 DNA polymerase, LIC-qualified Novagen 70099-3
DTT 100 mM homemade
Urea 2 M homemade
EDTA 500 mM pH 8.0 Homemade
LB broth (Miller) 500 g Athena ES 103

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