Protein Production
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.
Key equipment
- Biometra TAdvanced Twin PCR Thermal Cycler (Analytik Jena)
- Azure 300 Imaging System (Azure Biosystems)
- New Brunswick Innova® 44R Incubator Shaker (Eppendorf)
- Ecotron Incubator Shaker (INFORS HT)
- Biohazard box SafeFAST Classic S/D (class II, FASTER)
- New Brunswick Innova® 40/40R Benchtop Orbital Shaker (Eppendorf)
- PHCbi IncuSafe CO₂ static incubator (MCO-170AIC, PHC)
- New Brunswick S41i - CO₂ Incubator Shaker (Eppendorf)
- CellDrop FL Unlimited cell counter (DeNovix)
- Nikon ECLIPSE Ts2 FL inverted microscope (Nikon)
- Q700 Sonicator (QSONICA)
- Avanti J-26S XP High-Speed centrifuge (Beckman Coulter)
- ÄKTA start protein purification system (Cytiva)
- NGC Chromatography System (Bio-Rad)
- Trans-Blot® Turbo™ Transfer System (Bio-Rad)
- DS-11+ Spectrophotometer (DeNovix)
Expertise
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)
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 (HisTrap HP, Ni-INDIGO, Strep-Tactin®XT, GSTrap)
- Ion-exchange chromatography
- Size exclusion chromatography
- Protein concentration and dialysis
- Tag removal (TEV, SUMO, 3C protease)
Optimization of expression and purification protocols
Open-access
Providing full cloning services, recombinant protein production. Assistance with project design.
Core Facility contact
Miroslava Alblova – miroslava.alblova@ibt.cas.cz
More information about Protein Production and related services you can find in document Equipement and services of CIISB (page 31).
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