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About Protein Production, Strasbourg, France

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Sample preparation is not only a key issue for structural and molecular biology studies, but concerns life sciences development as a whole. Instruct centre France 1 offers state-of-the-art infrastructures for the expression and production of biomolecules and their complexes in prokaryotic and eukaryotic system.

Instruments Available:

IGBMC Strasbourg bacterial expression platform combines (i) a large set of expression vectors for Escherichia coli and (ii) robotized protocols, which enable a broad screening of soluble protein expression through the use of multiple fusion proteins and various growth conditions. IGBMC Strasbourg bacterial platform has also a long standing and unique expertise in protein complexes reconstitution and production by multi-expression in Escherichia coli. This technology is fully compatible with the automated protocols used for single expression. All bacterial expression vectors provided by the platform are compatible with major cloning strategies (e.g. restriction, Gateway, SLIC).

The insect cells expression platform at IGBMCThis facility is dedicated to protein and multi-protein complex production in insect cells using the baculovirus expression system for structural biology as well as for functional studies. It includes streamlined procedures for (i) the generation of recombinant baculoviruses (transfections, co-transfections) and amplification of a high titer viral stock, (ii) evaluation of recombinant baculoviruses and expression optimization using adherent cells or suspensions and (iii) protein expression in various scales and formats including production of multi-protein complexes.

IGBMC-CERBM Strasbourg provides expression in mammalian cells.Over expression of proteins in mammalian cells (hamster BHK21 cells) is achieved using an attenuated vaccinia virus vector (Modified Vaccinia Virus Ankara-MVA) that is user safe and may be handled under BSL1 conditions. Genes of interest are cloned downstream of a bacteriophage T7 promoter in a first step to check for feasibility. Protein expression is then examined after plasmid transfection into BHK21 cells and co-infection with a specialized viral vector encoding the bacteriophage T7 RNA polymerase.

For purification and subsequent research purposes, the protein has to be brought into solution by breaking the tissue or cells containing it. Several methods of cell lysis are commonly used to achieve this : mechanical disruption, sonication using high frequency sound waves, liquid homogenization and freeze/thaw cycles. The choice of the lysis method depends on the type of cells (bacteria, yeast, mammalian cells, etc), the volume of cell suspension and the fragility of the proteins to be recovered. The composition of the lysis buffer is also crucial to maintain the proteins of interest in the soluble fraction after cell breakage.

User Guide

Bacterial Expression

The Institute of Genetics and Molecular and Cellular Biology (IGBMC) Strasbourg bacterial expression platform combines a large set of expression vectors for Escherichia coli and robotised protocols, which enable a broad screening of soluble protein expression through the use of multiple fusion proteins and various growth conditions.

IGBMC bacterial expression platform offers two modes of access:

  1. The platform provides the expression vectors to the external users who perform cloning at their home institutions and come to IGBMC to perform the expression assays.
  2. The external users perform cloning and expression assays at the IGBMC platform.

Support includes training and supervision, as well as help with data analysis which is especially crucial for complex reconstitution.

Insect cells Expression

This facility is dedicated to protein and multi-protein complex production in insect cells using the baculovirus expression system for structural biology as well as for functional studies. It includes streamlined procedures for the generation of recombinant baculoviruses (transfections, co-transfections) and amplification of a high titre viral stock, evaluation of recombinant baculoviruses and expression optimisation using adherent cells or suspensions and protein expression in various scales and formats including production of multi-protein complexes.

Two modes of access with typical duration of stays ranging from a single to several weeks, depending on the project:

  1. The platform provides the transfer vectors to users who generate constructs in their home institutions and come to IGBMC to produce viruses and perform the expression assays.
  2. The external users perform cloning and expression assays at the IGBMC platform.

Support includes training and supervision in all aspects of the production run, including construct design and choice of purification strategies for reconstitution and purification of multi-protein complexes.

Mammalian Expression

Over expression of proteins in mammalian cells (hamster BHK21 cells) is achieved using an attenuated vaccinia virus vector (Modified Vaccinia Virus Ankara-MVA) that is user safe and may be handled under BSL1 conditions. Genes of interest are cloned downstream of a bacteriophage T7 promoter in a first step to check for feasibility. Protein expression is then examined after plasmid transfection into BHK21 cells and co-infection with a specialized viral vector encoding the bacteriophage T7 RNA polymerase. The IGBMC facility perform this feasibility step with any gene of interest cloned downstream of a T7 promoter and acquire results within a few days.

Support includes small scale expression tests with plasmids provided by users (needs the T7 promoter). Easy gene assembly using the “Biobrick” system based on compatible restriction sites (EcoRI, XbaI, SpeI, PstI) as described in (Ho-Shing et al., 2012) with a variety of N-terminal tags in a Gateway  entry vector for subsequent construction of recombinants virus is available. Participation of users is encouraged for training in the isolation of viral expression vectors and protein production runs at the IGBMC. Users will be provided with specialised materials and the most recent methods available.

Instruct Centre

Instruct Centre FR1

IGBMC

1, rue Laurent Fries

Illkirch

Strasbourg

France

www.igbmc.fr

Protein Production, Strasbourg, France

Contacts:

Marie-Christine Poterszman
Marie-Christine Poterszman
CNRS
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Marc Ruff
Marc Ruff
IGBMC-CERBM
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Arnaud Poterszman
Arnaud Poterszman
CNRS
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Catherine Birck
Catherine Birck
IGBMC-CERBM
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