Laboratory of Protein Engineering

 


 

Head:
Anna Urbanowicz, PhD

 

 


Staff:
Associate Prof. Michał M. Sikorski DSc Marta Grzechowiak PhD Eng, Joanna Śliwiak MSc, Alina Kasperska MSc,  Marian Gawron PhD, Dr. Jakub Barciszewski

 

 


PhD Students:
Paulina Bierwagen MSc Eng.

 

 


 

Rys.1

 


 


 

 

 

 

 

Key words:

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

 

Research offer:

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:

Equipment available in the Laboratory can be used for the following purposes :

 

1.  Construction of plasmids for protein expression:

- thermocycler with dual gradient blocks,

- incubators

- microcentrifuges,

- thermoblock,

- 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

 

Important:

  • New users are asked to contact with the Head of the Laboratory.
  • Before using the equipment please make a reservation.
  • Users of Laboratory equipment prepare the necessary reagents to perform their own research, purchase their own essential accessories that are consumed during the use of equipment and cover the cost of repairing the damage made by them (e.g. breakage of the cuvette, etc.). 
  • Users which plan a regular use of the equipment or the involvement of employees in their research are asked to contact the Head of the Laboratory to discuss the principles of cooperation.

 

Scientific profile:

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

  • Crystallographic and biophysical studies on the interactions between pathogenesis-related proteins class 10 with flavonoids, melatonin and melatonin derivatives.
  • Structural studies of transcription factor WRKY50 from A. thaliana, a positive regulator  of the abscisic acid and repressor of the jasmonic acid signaling pathways.
  • Structural studies of proteins involved in A. thaliana phosphate metabolism: apyrases hydrolyzing diphosphate from NTPs and inorganic pyrophosphatases hydrolyzing pyrophosphate to phosphate used then for plant metabolism and biosynthesis of energy-rich molecules (recycling of the phosphorus in plant cells). 
  • Structural studies and studies of interactions between the selected proteins from Ixodes ticks and Borrelia spirochetes, aimed at understanding of the molecular bases of processes associated with the spread and prevention of Lyme disease.
  • Studies of the relationships between the structure of the viral capsid and the conditions of surrounding solution using a model plant ssRNA virus Brome mosaic virus (BMV).

 

Most important research achievements:

  • The discovery of the disorder nature of  TROSPA protein from Ixodes ricinus.
  • Refinement the first crystal structure of the plant inorganic pyrophosphatase AtPPA1 from A. thaliana (PDB code:  4lug)
  • Analysis of the AtPPA1 mutants activity: D98N, D103N AND D135N.
  • Refinement and analysis of modulated superstructure of the complex of Hyp-1 protein from St. John’s wort with fluorescent probe ANS, which contains 28 protein copies in asymmetric unit (PDB code: 4n3e).
  • Determination of melatonin as a binding partner for PR-10 proteins after screening different phytohormones; solution of the first structure of plant protein (PR-10) in complex with melatonin (PDB code: 5i8f).

 

Actual research projects:

  • Structural studies of proteins crucial for tick-pathogen-host interactions (NSC project).
  • Physicochemical characterization of plant protein-phytohormone interactions (statutory project).  

 

Selected publications:

  1. Śliwiak, J., Dauter, Z. & Jaskólski, M. (2016). Crystal structure of Hyp-1, a Hypericum perforatum PR-10 protein, in complex with melatonin. Front. Plant Sci. 7, a.668.
  2. Urbanowicz, A., Lewandowski, D., Szpotkowski, K., & Figlerowicz, M. (2016). Tick receptor for outer surface protein A from Ixodes ricinus - the first intrinsically disordered protein involved in vector-microbe recognition. Sci Rep. 6, 25205. doi: 10.1038/srep25205.
  3. Śliwiak, J., Dolot, R., Michalska, K., Szpotkowski, K., Bujacz, G., Sikorski, M. & Jaskólski, M. (2016). Crystallographic and CD probing of ligand-induced conformational changes in a plant PR-10 protein. J. Struct. Biol. 193, 55-66.
  4. Śliwiak, J., Dauter, Z., Kowiel, M., McCoy, A. J., Read, R. J. & Jaskólski, M. (2015). ANS complex of St John’s wort PR-10 protein with 28 copies in the asymmetric unit: a fiendish combination of pseudosymmetry with tetartohedral twinning. Acta Crystallogr. D71, 829-843.
  5. Ruszkowski, M., Śliwiak, J., Ciesielska, A., Barciszewski, J., Sikorski, M. & Jaskólski, M. (2014). Specific binding of gibberellic acid by Cytokinin-Specific Binding Proteins: a new aspect of plant hormone-binding proteins with the PR-10 fold. Acta Crystallogr. D70, 2032-2041.
  6. Śliwiak, J., Jaskólski, M., Dauter, Z., McCoy, A. J. & Read, R. J. (2014). Likelihood-based molecular-replacement solution for a highly pathological crystal with tetartohedral twinning and sevenfold translational noncrystallographic symmetry. Acta Crystallogr. D70, 471-480.
  7. Śliwiak, J., Dauter, Z. & Jaskólski, M. (2013). Hyp-1 protein from St John’s wort as a PR-10 protein. BioTechnologia 94, 47-50.