Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Lakshmi Sumitra Vijayachandran, Cristina Viola, Frederic Garzoni, Simon Trowitzsch, Christoph Bieniossek, Maxime Chaillet, Christiane Schaffitzel, Didier Busso, Christophe Romier, Arnaud Poterszman, Timothy J Richmond, Imre Berger

Research output: Contribution to journalArticle (Academic Journal)peer-review

67 Citations (Scopus)

Abstract

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

Original languageEnglish
Pages (from-to)198-208
Number of pages11
JournalJournal of Structural Biology
Volume175
Issue number2
DOIs
Publication statusPublished - Aug 2011

Bibliographical note

Copyright © 2011 Elsevier Inc. All rights reserved.

Keywords

  • Academies and Institutes
  • Animals
  • Automation, Laboratory
  • Baculoviridae
  • Cells, Cultured
  • Cloning, Molecular
  • Escherichia coli
  • Europe
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins
  • Multiprotein Complexes
  • Polyproteins
  • Protein Engineering
  • Recombinant Proteins
  • Spodoptera

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