Core facility of biotechnology of recombinant proteins
Production of recombinant proteins by the choice of appropriate expression system, optimization of the cultivation conditions of producer cells and elaboration of purification protocols. Mostly E.coli expression system is used for immunologically relevant proteins and virus-like particles (VLPs), but also yeast expression systems have been used in BMC.
Cultivation of cells
For preparative cultivation of bacterial or yeast cells three-module Multitron (Infors, Switzerland) shakers (two units) are generally used for controlled conditions. For analytical purposes thermal room with shakers for flasks and tubes are normally used. In situ sterilized BioFlo 410 Benchtop Fermentor (New Brunswick Scientific Co, USA) is available for cultivation of up to 10 L of bacterial or yeast culture. Media are prepared by the use of Systec V-150 (Systec GmbH, Germany) autoclaves (two units).
It is planned to purchase a number of necessary equipment for controlling of biotechnological processes: digital controlled and monitorable 4-module laboratory bioreactor unit EDF 1.1, exhaust gas (O2 and CO2) analyzer BlueInOne, compatible equipment for existing HPLC equipment (Shimadzu Prrominence LC-20) for substrate analysis (glucose, acetate etc).
Harvesting and disintegration of biomass
For analytical purposes cells are harvested using Eppendorf (Eppendorf AG) analytical centrifuges. For preparative amounts Beckman J2-21 (Beckman Coulter, USA), Eppendorf 5810 R as well as K26D (VEB Zentrifugenbau Engelsdorf, DDR) are used. Ultrasonic disintegrators Soniprep 150 (MSE, UK) and UD-20 (Techpan, Poland) are used for analytical/preparative and FRENCH Press Cell Disrupter (Thermo Electron Corporation, USA) for preparative disruption of biomass.
For scale-up purposes it is planned to buy more productive and powerful cell disintegration system – Microfluidizer LM 20.
Purification of proteins
The following equipment for protein purification from GE Healthcare Life Sciences are in use in our facility: lab-scale chromatography system Äkta prime plus (two units), lab-scale chromatography system ÄKTA Avant (with Unicorn 6.2 software), lab-scale chromatography system Äkta Pure 25 L (with Unicorn 6.3 software). Unichromat 1500 (UniEquip, Germany) cabinet is available for purification at controlled temperature. Different strategies are used in purification of proteins depending on the particular task: for purification of VLPs gel-filtration on Sepharose CL2B, 4B, 6B, 4FF, 6FF, Superose 6, Superdex 75, Superdex 200, and ion exchange chromatography using anionic carriers Sepharose Q HP, Fractogel DEAE, Source 15Q and cationic carriers Sepharose SP HP, Source 15SP are used. Imobilized metal-affinity chromatography (IMAC) is used for His-tag proteins and hydrophobic interaction chromatography (HIC), reverse phase chromatography (using media with PharmPrep HP 100 RP-18 and Lichroprep RP-18, 25-40µm) as well as multimodular chromatography media (hydroxyapatite) are used in specific cases in complex purification protocols.
For scale-up purposes it is planned to buy tangential crossflow filtration equipment AKTA flux S with a set of hollow fibre cartridges (different cut of values and different productivity).
Process control and characterization of particles
Modular analytical Prominence HPLC system (Shimadzu, North America) is used for the control of purification process and characterization of end products of purification. DLS (Dynamic Light Scattering) measurements are performed using Malvern Zetasizer Nano ZS equipment (Malvern Instruments, UK) for characterization of particle size and homogeneity of protein samples.
Ivars Petrovskis, Dr.biol
cultivation of recombinant proteins, purification of recombinant proteins
Jānis Bogans, MSc.Chem
Jeļena Žakova, Msc.Biol
experiments with recombinant cultures and proteins