The MultiBac Protein Complex Production Platform at the EMBL

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 relevant constructs in silico and validate strategy thoroughly before proceeding to experimental work. For example, genes of interest should be ....

Results

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

Discussion

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 .......

Materials

List of materials used in this article

Name	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