Core facility of recombinant protein biotechnology
Suitable expression systems, optimisation of production conditions and different purification protocols are used to produce recombinant proteins. Different E. coli and yeast expression systems are used to produce immunologically significant proteins and virus-like particles (VLP)
Three-module shakers Multitron II (Infors, Switzerland) are used to cultivate bacterial and yeast cells under controlled conditions. In situ sterilizable BioFlo 410 enzyme (New Brunswick Scientific Co, USA) is available for the cultivation of E. coli and yeasts in volumes of 2 to 10 litres. Culture media are prepared using autoclaves Systec V-150 (Systec GmbH, Germany).
Digitally controlled 4-module laboratory bioreactor EDF 1.1, outlet gas (O2 and CO2) analyzer BlueInOne, and equipment for the analysis of substrates (glucose, acetate, etc.) with HPLC machine Shimadzu prominence LC-20 are available to control biotechnological processes.
Biomass collection and disruption
For analytical purposes, biomass is collected with Eppendorf analytical centrifuges. For larger quantities, Beckman J2-21 and AVANTI J-301 (Beckman Coulter, USA), Eppendorf 5810 R centrifuges are used.
The Soniprep 150 (MSE, AK) and UD-20 (Techpan, Poland) ultrasound disintegrators are used for analytical/preparative biomass disintegration, while the FRENCH Press cell disrupter (Thermo Electron Corporation, USA) machine is used for preparative biomass disintegration. Powerful disintegration machine Microfluidizer LM 20 (Microfluidics, Canada) is available for scaling-up purposes.
Purification of proteins
Following machines from GE Healthcare Life Sciences are used for protein purification: laboratory scale chromatography systems ÄKTA prima plus, laboratory scale chromatography system ÄKTA Avant (with Unicorn 6.2 software), laboratory scale chromatography system ÄKTA pure 25L (with Unicorn 6.3 software). The Unichromat 1500 machine (UniEquip, Germany) is available for the work with thermolabile proteins.
Depending on the protein, different purification strategies are available: gel filtration on Sepharose CL2B, 4B, 6B, 4FF, 6FF, Superose 6, Superdex 75, Superdex 200 carriers, ion exchange chromatography using Sepharose Q HP, Fractogel DEW, Source 15Q and Cathionites Sepharose SP HP, Source 15SP. His-tag proteins use immobilized methal-affinity chromatography (IMAC). For special cases, complex purification protocols can be elaborated using hydrophobic interaction chromatography (HIC), reverse phase chromatography (PharmPrep HP 100 RP-18 and Lichroprep RP-18, 25-40 μm), as well as multi-modular chromatography carriers (hydroxyapatite).
Ultracentrifuges L-100XP and TL-100 with different rotors (Beckman) are used to purify viruses and nanoparticles.
Process control and product characterization
A modular analytical prominence HPLC system LC20 (Shimadzu, USA) is used to control purification processes and characterize the purified product. Dynamic light diffusion (DLS) measurements for assessing particle size and protein homogeneity shall be performed with the Malvern Zetasizer Nano ZS equipment (Malvern instrument, UK).
Andris Zeltiņš, PhD
Head of the core facility , senior researcher
- Development and optimization of cultivation protocols for producers of recombinant proteins
- Development and optimization of recombinant protein purification protocols
- Application services for centrifuges and ultracentrifuges
- Contract studies
- Consultancy for scientific personnel and industry in obtaining recombinant proteins