The IMPReSs facility offers a panel of services dedicated to the production, purification and characterization of membrane proteins. Different expression systems are routinely used for the expression of membrane but also cytosoluble proteins: the yeast Pichia pastoris and the bacterial system Escherichia coli are mainly employed, other organisms such as mammalian cell lines, Pseudomonas aeruginosa and Anabaena sp. PCC 7120 being also available.
In order to fit your specific needs, IMPReSs offers modulable and flexible services from molecular cloning steps to purification and characterization of the protein of interest.
1. Consulting on production strategies
We bring our expertise to set up with you the best cloning, production, purification and characterization strategies, in the light of your requirements and specifications.
Molecular cloning of a gene of interest can be done either from a plasmidic DNA or a cDNA provided by the client, or from a synthetic gene ordered from an external provider. A large panel of expression vectors dedicated to each expression system are available at IMPReSs. They can be customized according to your needs (affinity tags, secretion signals, spacers, cleavage sequences…). Conventional cloning techniques are used to obtain the desired recombinant vectors.
3. Expression-level screening assay
As a first step for every new target, we propose a feasibility study for the production of the protein of interest in the best-suited expression system(s).
For the Pichia pastoris expression system, the original Yeastern Blot technique that we developed in the lab allows high-throughput screening of the level of expression of recombinant clones in the 96-wells plate format.
4. Production and preparation of protein samples
The production step of the protein can be performed in shake flasks up to a volume of 10 liters of medium, according to your needs. A scale-up in a 3-liters fermenter can also be considered upon request.
Membrane proteins can be provided in the form of membrane preparations, obtained by differential ultracentrifugation after cell lysis. Several extraction and solubilisation conditions can also be investigated for the best compromise between extraction yield and protein quality.
Five automated purification devices (Äkta Pure, Äkta Purifier and Äkta FPLC) are available at IMPReSs, as well as a complete panel of affinity, ion exchange and size exclusion chromatography columns and supports. Depending on your needs, more specific methods, such as chromatofocusing, can also be used for the purification step.
In the case of membrane proteins, extensive screenings of the purification conditions are frequently performed in order to best adapt to each protein (buffer composition, screening of detergents, exchange of detergents, protein processing, chromatography methods...).
Different biochemical analysis are used for the characterization of the protein sample at each step of the process (electrophoresis, immunodetection, size exclusion chromatography). Mass spectrometry analyses can also be planned.
In addition, when available, activity assays can be performed at each step of the production process for an advanced characterization of membrane proteins. IMPReSs is notably fully equiped and accredited to performed radioactive ligand binding and transport assays, including at the 96-wells plate format.
7. Library of GPCR-expressing yeast strains
The IMPReSs facility owns a library of Pichia pastoris strains expressing almost a hundred different G protein coupled receptors (GPCRs) fused to different affinity tags. Their production and, for some of them, their purification are available upon demand.