Advancing Epigenetics Towards Systems Biology

The MultiBac BEVS for producing proteins and their complexes (prot 54)

Frederic Garzoni, Christoph Bieniossek, Imre Berger

Introduction

Protein complexes are central to cellular function. Many important complexes in eukaryotes require recombinant overproduction for a detailed analysis of their structure and function. Baculovirus expression vector systems (BEVS) have become increasingly popular for the production of such specimens, in particular for complexes which depend on a eukaryotic host cell machinery for proper folding, post-translational modification, authentic processing and correct targeting to cell compartments for their activity. The MultiBac system (Fig. 1) is a BEVS specifically designed for producing large eukaryotic complexes with many subunits [Berger et al., Nat. Biotechnol. 2004, Fitzgerald et al., Nat Methods, 2006]. It consists of an array of plasmids which facilitate multigene assembly, a modified baculovirus genome that with optimized protein production properties, and a set of protocols detailing every step from inserting encoding DNAs in the plasmid array to protein production by this technology. The components of the system and the protocols used are continuously being improved, developed and streamlined to simplify handling and improve efficacy [Trowitzsch et al., J. Struct. Biol. 2010, Vijayachandran et al., J. Struct. Biol. 2011]. We believe that our efforts have reduced the previously perceived complexity of the baculovirus/insect cell system to the level of protein expression in E. coli. Detailed protocols are available for utilizing the MultiBac system [Fitzgerald et al., Nat. Methods 2006; Bieniossek et al., Curr. Protoc. Prot. Sci. 2008], although certain details have been altered over the years and are bound to be refined in the future. In this protocol, we present the protocol as it is currently in operation at the MultiBac platform of Eukaryotic Expression Facility (EEF) at the EMBL Grenoble (Fig. 2), as part of the trans-national access scheme in the P-CUBE (www.p-cube.eu) and BioSTRUCT-X (www.biostruct-x.eu) projects of the European Commission, Framework Program 7. The protocol has been designed such that it can be performed successfully by scientists with or without previous knowledge of or experience with BEVS for expressing their targets during a ten working-day visit at our MultiBac platform.
Parts of the protocols are specific for MultiBac (plasmid fusions, multigene generation, fluorescence tracking). However, all virus amplification and protein production protocols are generically applicable to other baculovirus systems that utilize recombinant bacmids (for example Bac-to-Bac, Invitrogen).

PDF version

Frederic Garzoni, Christoph Bieniossek, Imre Berger

EMBL Grenoble Outstation and Unit of Virus Host Cell Interactions UVHCI (UMR5322)
6 Rue Jules Horowitz, 38042 Grenoble CEDEX 9, FRANCE

Corresponding author: Frederic Garzoni
Email feedback to: This email address is being protected from spambots. You need JavaScript enabled to view it.

Frederic Garzoni, Christoph Bieniossek, Imre Berger
Mon, Jan 21st 2019- Fri, Jan 25th 2019

This meeting will be the first digital health meeting that focuses specifically on the scientific foundations and the health applications of digital technologies. Taking a novel sensor or new device...

Wed, Jan 30th 2019- Thu, Jan 31st 2019

"Conferences Series LLC LTD is organizing “2nd World Congress on Petrochemistry” scheduled to be held during January 30-31,2019, Bangkok, Thailand. The conference invites all the participants across t...

Mon, Feb 25th 2019- Tue, Feb 26th 2019

The seminar will feature approximately 10 talks and 2 poster sessions. All attendees are expected to actively participate in the conference, either by giving an oral presentation or presenting a poste...

Mon, Mar 11th 2019- Tue, Mar 12th 2019

We are pleased to invite you to the upcoming conference 12th World Congress on Cell and Tissue Science scheduled in Singapore on March 11-12,2019. It will bring world-class personalities and researche...

  • Holiday inn Singapore Atrium 317 Outram Road Singapore 169075
  • Organizer

LAST EVENTS

EpiGeneSys Final
Meeting in Paris

Thur. 11 February 2016 - Sat. 13 February 2016

More than 280 scientists attended the fifth Annual Meeting of EpiGeneSys. The conference kicked off with a talk by coordinator Geneviève Almouzni, Director of the Research Center at the Institut Curie, highlighting the achievements of the network over more than five years...

Maison des océans - Paris Read more

PAST EVENTS

The Non-Coding Genome ...

December 3-4 th, 2015

The last training workshop of the EpiGeneSys network

Hotel Mediterraneo - Rome, Italy Read more

Paris / TriRhena Chromatin Club

July 9th, 2015

...exciting talks and network with members of the Chromatin community!

... An EpiGeneSys TAB workshop

June 11st-12nd , 2015

... learn about current approaches to single cell epigenetics and to meet up and network with...

Montpellier, FranceRead more

Latest publications

2017-06-27

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Read more
2017-05-26

Proliferation Drives Aging-Related Functional Decline in a Subpopulation of the Hematopoietic Stem Cell Compartment.

Read more
2017-04-30

The impact of rare and low-frequency genetic variants in common disease.

Read more