Introduction
The AFMB service facility dedicated to quantitative production of recombinant eukaryotic proteins is labeled by the French Scientific Group of Interest for Health Biology and Agronomy (IBISA). It is also one of the five leading facilities of the French Infrastructure for Integrated Structural Biology (FRISBI). Each year, the facility generates several eukaryotic proteins for research projects involving structural studies and protein-protein, protein-DNA or protein-ligand interaction analyses.
Strategy
The facility staff members and internal users analyze the feasibility of projects submitted by external users, select potentially well-adapted expression systems (choice of cell line, expression vector, tag) and experimental conditions (culture medium composition, time course of expression) for efficient production, and assay them at the analytical scale.
If the protein expression level is sufficiently high, the cell culture volume is directly up-scaled to the project needs.
If the expression level is low, a clonal (mammalian) or semi-clonal (insect) cell line is generated within a few weeks and then either maintained into production or stored frozen for later production.
 |
 |
 |
Technology
The latest technologies are used for production of soluble or membrane-linked or -embedded eukaryotic proteins. The facility staff works in close relationship with the users, who keep taking care of the detection, purification and characterization steps associated with their protein.
Production
Production in mammalian cells
The AFMB lab owns an operating license for patent WO2009/137911 [1], which allows the facility to use HEK293-6E cells (EBNA, Epstein-barr virus antigen-1) along with dedicated oriP vectors (PYD, pTT). Compared with more classical systems, this new and innovative technology warrants higher yields of transient protein production.
HEK293-GNT1- cells [2] can be also used for production of N-glycosylated proteins. These cells achieve simple and homogeneous N-glycosylation patterns (Man5GlcNAc2) likely to favor crystallization endeavors.
Other cell lines such as COS-7 or CHO-K1 cells can be also used if required.
Stable cell lines can be also established within a few months if need by using the tetracyclin-inducible system, T-REx, from Life Technologies (CHO-TREx or HEK-TREx) [3].
The proteins are produced in Multi-Flasks, Spinner flasks or Roller bottles depending on the yields and amounts required by the users.
Production in insect cells
The proteins are produced from drosophila S2 cells transfected with metal-inducible vectors (pMT-DEST48, pMT/BiP/V5-His, Life Technologies) [4]. Polyclonal or pseudo-clonal stable cell lines can be established within a few weeks.
The proteins can also be produced from Spodoptera frugiperda sf9 cells infected with a baculovirus (Bac-toBac system, Life Technologies) [5]. After the initial step of recombinant bacmid transfection a viral stock is generated, titrated, and used to infect those cells that present an optimal MOI (Multiplicity Of Infection).
The proteins are produced in Multi-Flasks (adherent cells) or Spinner flasks (suspension cells) depending on the yields and amounts required for the project.
Detection and quantification of the produced proteins
The proteins secreted in the culture medium are directly quantified using a dot-blot assay and an Octet Red96 (ForteBio) apparatus, and their proper molecular masses are verified by Western blot.
The membrane-linked or membrane-embedded proteins are also quantified after detergent-solubilization steps.
Material
4 CO2 incubators (Thermo Heracell 240i)
3 thermoregulated storage cabinets
4 laminar flow hoods
1 epifluorescent microscope (Nikon Eclipse TS100)
1 inverted microscope (Olympus CK2)
2 centrifuges et 1 ultracentrifuge (Avanti J-26XP)
1 Octet Red96 apparatus (ForteBio)
Dot-blot and Western-blot apparatus
