Laboratory of Protein Engineering
Ixodes ticks proteins, Borrelia spirochetes proteins, Lyme desease, PR-10 protein complexes, phytohormones, melatonin, transcryption factors, WRKY, A. thaliana plant pyrophosphatase, AtPPA1, isothermal titration calorymetry, iTC, dynamic light scattering, DLS, static light scattering, SLS, micro-scale thermophoresis, MST
Our laboratory is equipped with the unique devices for production and physicochemical characterization of recombinant proteins produced in the bacterial system (E. coli).
Fulfilling the scientific mission we provide the collected equipment to all interested researchers involved in studies of structure and biological function of proteins. We also offer an expertise in expression, purification and protein preparations quality assessment for further use to determine their structure and examine biological activity.
The research currently being carried out in the Laboratory are mainly characterization of the protein-protein, protein ligand-interactions and crystallization of these complexes for structural studies (X-ray crystallography).
Equipment available in the Laboratory can be used for the following purposes :
1. Construction of plasmids for protein expression:
- thermocycler with dual gradient blocks,
- chambers for agarose gel electrophoresis
Quality of plasmid constructs harboring protein coding sequences is checked by DNA sequencing service outside the Institute.
2. Overproduction of recombinant protein in E. coli system:
- incubator shaker with adjusted temperature from 5 to 50ºC,
- sonicator with two interchangeable probes (according to sample volume),
- preparative centrifuge (rotors available: 8 x 50 ml and 6 x 500 ml).
3. Protein purification:
- apparatus for protein electrophoresis at denaturing conditions (SDS/PAGE)
- vacuum set for chelating chromatography (Ni-NTA Sepharose resin),
- FPLC system (ÄKTA) for column chromatography of proteins (preparative columns for: SE – size exclusion, IEC – ion exchange and AC – affinity chromatograqphy),
- possibility of preliminary purification of proteins from crude extracts by ammonium sulfate precipitation, dialysis and ion exchange chromatography on DE52-cellulose column.
4. Physicochemical analysis of recombinant proteins:
- size exclusion chromatography (FPLC) – oligomers formation
- dynamic light scattering (DLS) and static light scattering (SLS) – measurement of size and molecular weight of proteins and polymers
- determination of binding constans of ligands and stoichiometry of protein to ligand in complexes applying isothermal titration calorymetry (iTC) and/or microscale thermophoresis (MST) methods
5. Protein crystallization
- development of crystallization conditions using commercial screens of precipitation agents
with use of crystallization robot
- crystallization of proteins/protein complexes by the hanging drop vapor diffusion technique – grooving crystals for diffraction data collection
6. Diffraction data collection
- collection high resolution diffraction data in synchrotrons: DESY, BESSY (Hamburg, Berlin - Germany) or in Lund Synchrotron (Sweden)
- diffraction data collections at synchrotrons are carried out only by trained personnel of the Crystallography Department – CBB
7. Laboratory is also equipped with:
- deep frezer (-80ºC)
- multi-modular water purification system
Laboratory of Protein Engineering was established in 2013, as a consequence of purchasing a modern research equipment for study protein structure and biological function, extracted from EU funds for the project in frame of the Innovative Economy etittled:. "Increasing the research potential of the Institute of Bioorganic Chemistry in the structural analysis of biomolecules by NMR and crystallography."
The research currently held by us involves both animal and plant proteins: plant pathogenesis-related proteins class 10 (PR-10), plant transcription factors and apyrases and proteins crucial for the interaction between Ixodes ticks and Borrelia spirochetes causing Lyme disease. These studies are carried out with the use of recombinant proteins produced in the bacterial system and focus on physico-chemical characteristics of protein-protein and protein-ligand interactions, structural studies of these complexes by their crystallization and solving structures, as well as the structural studies of proteins in solution.
Current research activity:
Most important research achievements:
Actual research projects: