Protein Production, Vestec near Prague, Czech Republic

The core facility for protein production provides comprehensive services, covering every step from DNA to purified protein. These include gene cloning into expression vectors using various techniques and heterologous expression in Escherichia coli for bacterial expression, HEK293T, Expi293, and Expi293F™ GnTI- for mammalian expression, and the baculoviral system in Sf9 or High Five™ insect cells. The expression is followed by multistep protein purification.

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User Guide

Cloning services
- Design of expression vectors
- DNA template control by whole plasmid sequencing
- Ordering gene fragments from an external company
- Cloning using traditional restriction enzymes
- Restriction-free methodologies (RF, KLD, In-Fusion)
- Site-directed mutagenesis
- Plasmid DNA preparation (minipreps, midipreps, maxipreps)
- Preparation of competent cells
Recombinant protein production in E. coli
- Small-scale expression and solubility tests in various E. coli strains
- Large-scale protein expression
- Protein production in minimal media
Recombinant protein production in insect cells
- BacPAK™ (TaKaRa) and FlexiBAC baculovirus systems
- Small-scale cell transfection and expression verification by Western blot
- Baculovirus amplification
- Large-scale production in Sf9 or High Five™ cells
Recombinant protein production in mammalian expression systems
- Expression in HEK293T/17SF, Expi293™, Expi293F™ GnTI- cells
- Small-scale cell transfection and expression verification by Western blot
- Large-scale cell transfection and protein production
Recombinant protein purification
- Affinity chromatography (HiTrap HP, Ni-INDIGO, Strep-Tactin®XT, GST, cobalt)
- Ion-exchange chromatography
- Size exclusion chromatography
- Protein concentration and dialysis
- Tag removal (TEV, SUMO, 3C protease)
Optimization of expression and purification protocols