Ewa Szołajska, PhD, DScCell Culture and Protein Production Facility
Service and research scope
Our facility provides to IBB researchers and PhD students access to BSL2 laboratory space and basic equipment that are required for research that involves mammalian cell cultures and flow cytometry. We also offer services for protein production in baculovirus-insect cell expression systems that generate posttranslationally modified recombinant proteins with a native conformation and biological function.
Ewa Szołajska (PhD, DSc), email:email@example.com, tel.: 22 592 24 20
Elżbieta Speina* (PhD, DSc), email: firstname.lastname@example.org, tel.: 22 592 33 32
*person responsible for flow cytometer
Our services include the production of recombinant proteins in a baculovirus-insect cell system. The following recombinant proteins were used for the implementation of several projects that were performed at the Institute:
- prokaryotic: dioxygenase AlkA of Pseudomonas putida.
- human: RIO1 and RIO3 kinases, catalytic domain of Janus kinase 2 (Jak2), checkpoint kinases 1 and 2 (Chk1, Chk2), signal transducer and activator of transcription 5A (STAT5A), model of CUB2 domain, fat mass and obesity‐associated protein (FTO), calmodulin.
- viral: penton base protein (Pb) of human adenovirus (Ad; self-assembles into virus-like particle (VLP)), Pb with inserted epitope derived from M1 influenza matrix protein or from ovalbumin (OVA), hemagglutinin (HA) of avian influenza A (H5N1) virus in fusion with WW domains and variant with trimerization domain and N-terminal fragment of Ad fiber protein.
For customers from other institutions, the following proteins were successfully produced:
- mouse: tubulin and kinesin Kif5a, including wildtype, mutated, and tagged protein variants.
- insect: kinesin-like ncd protein of Drosophila melanogaster.
- human: DNA topoisomerase type I and kinesin HSET.
Recombinant proteins were obtained with variable yields, but all were properly folded and functional. This was confirmed by structural analyses, enzyme activity tests, motility assays, and in vitro and in vivo immunological tests in the case of virus-like particle -based vaccine preparations.
Two training courses on techniques that are used for recombinant protein production in baculovirus-infected cells were conducted for employees of ADAMED (2010) and the Laboratory of Protein Structure at IIMCB (2011).
In addition to the production of proteins in the baculovirus expression system, the facility plans to support IBB researchers by offering in-house-produced reagents for the preparation of DNA libraries and DNA transfection and purification of recombinant proteins by affinity chromatography. In the future, we plan to implement cost-effective procedures for the expression of recombinant proteins in human 293 cells.
Our facility has supported several laboratories which research involves the following topics:
- Studies of molecular mechanisms of the immune response that underlies allergy to nickel and the analysis of copper concentrations in human 293 cells, tested with single-cell resolution (Prof. Wojciech Bal group).
- Studies of the influence of the overexpression of proteins of homologous recombination on the function of the CRISPR/Cas9 system in human 293 cells (Prof. Matthias Bochtler group).
- Analyses of the receptor for advanced glycation endproducts (RAGE) activation process in the presence of molecules that interact with the cell membrane of mammalian cells (Prof. Michał Dadlez group).
- Development of proteomic methods that support the diagnosis of breast and lung cancer (Prof. Michał Dadlez group).
- Studies of the role of a common subunit of RNA polymerases I and III in colorectal cancer (Dr. Damian Graczyk group).
- Studies of (i) the role of human homologues of the AlkB protein in DNA repair, RNA modification, genome stability, cell metabolism, and the development of obesity and type 2 diabetes and (ii) the anti-tumor activity of compounds from the stilbene, oxepin, and anthraquinone groups (Prof. Elżbieta Grzesiuk group).
- Studies of biological properties, including cytotoxic activity, of new dibenzoadiazocin derivatives (Elżbieta Grzesiuk and Prof. Wojciech Bal groups).
- Studies of the influence of recombinant influenza virus hemagglutinins, with high and low-mannose glycosylation on the proliferation of splenocytes and production of specific cytokines (supervised by Dr. Edyta Kopera).
- Studies of the role of posttranslational modifications in the regulation of human polymerase ι (Prof. Ewa Śledziewska-Gójska group).
- Identification of epigenetic mechanisms that regulate the expression of key tumor suppressors in clear cell kidney cancer (Dr. hab. Tomasz Sarnowski, Prof. of IBB PAS, group).
- The use of adenovirus dodecahedron as a vector for the intracellular delivery of therapeutic factors (Dr. hab. Ewa Szołajska group).
- Analysis of the content of polyisoprenoid alcohols, lipid derivatives, and oxidized forms in HeLa cells. Research on SRD5A3 polyprenol reductase in 293 and HeLa cells (Prof. Ewa Świeżewska group)
- Proteomic analyses of intracellular S-nitrosylation under physiological and pathological conditions (Prof. Michał Dadlez group).
- Studies of the influence of specific RNA on the level of proinflammatory proteins in human and murine fibroblasts (Dr. hab. Urszula Zielenkiewicz group).
Equipment for cultures of mammalian cells includes:
- 5 Class II biological safety cabinets:
BioWizard Silver SL-130 Blue Series (Kojar)
TOP Safe 1.5 and TOP Safe 1.2 (Bioair)
Steril CTH (Angelantoni Life Science) dedicated to handling cytotoxic drugs
Laminar flow hoods with activated carbon filter (Polon)
- 4 CO2 Incubators:
2 NU-5510 CO2 (NuAire)
Igo 150 Cell Life
Galaxy 48R (New Brunswick) with O2 control for culture under hypoxic conditions
- Shaking incubator Ecotron (Infors HT)
- CKX41 inverted microscope (Olympus) equipped with fluorescence illuminator, UIS2 filters (blue, green, ultraviolet), and 4´, 10´, 20´, and 40´ objectives
- BD FACSCalibur cell analyzer and sorter
2 lasers: 15 mW, 488 nm, air-cooled Argon-ion laser and second nominally 635 nm
4 photomultipliers with band pass filters: 530 nm (FITC), 585 nm (PE/PI), 661 nm (APC), and > 650 nm (PerCP) with base unit, > 670 nm (PerCP) with FL4 option
- BD FACSCalibur cell analyzer and sorter
- 5810R centrifuge (Eppendorf), refrigerated with a A-4-62 swingout rotor (with adapters for 15/50 ml conical tubes and plates) and fixed-angle rotor for 1.8 ml tubes
- Automated cell counters (Invitrogen)
- Water bath (PolyScience)
- MDF-U53V ultra-low temperature freezer (Sanyo)
- 5 Refrigerators/freezers
Equipment for protein production in baculovirus-insect cell systems includes:
- Incubators without control of gas concentrations:
- BD53 (Binder) for adherent cultures
- Shaking incubators: Multitron Pro and Ecotron (Infors HT)
- NU-437 400E class II biological safety cabinets (NuAire)
- Inverted microscope (Carl Zeiss Jena)
- AKTA FPLC System with UPC-900 monitor, P-920 Pump, and Frac-950 Fraction Collector (Amersham Biosciences)
- Jean-Pierre Andrieu, Institut of Structural Biology, Grenble, France
- Wim P. Burmeister, Université Grenoble Alpes, Unit of Virus Host Cell Interactions, Grenble, France
- Jadwiga Chroboczek, Université Grenoble Alpes
- Pascal Fender, Institute of Structural Biology, Grenoble, France, www.ibs.fr/research/research-groups/methods-and-electron-microscopy-group/fender-team/?lang=fr
- Jean-François Dufour, Department for BioMedical Research, University of Bern, Switzerland, dbmr.unibe.ch/research/research_groups/personenpool/hepatology/prof_dr_med_dufour_jean_franois/index_eng.html
- Jacek Jemielity, Laboratory of Bioorganic Chemistry, Centre of New Technologies, University of Warsaw, Poland, cent.uw.edu.pl/pl/laboratoria/laboratorium-chemii-bioorganicznej/#z1
- Joanna Kowalska, Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland
- Benedicte Lambrecht, Avian Virology and Immunology Service, Veterinary and Agrochemical Research Centre, CODA-CERVA, Brussel, Belgium
- David Laurin, Etablissement Français du Sang Auvergne Rhône-Alpes, Grenoble, France
- Remigiusz Worch, Laboratory of Biological Physics, Institute of Physics Polish Academy of Sciences, Warsaw, Poland, ifpan.edu.pl/SL-4/index.php?group=remiwo
- Tetsuya Nakatsura, Division of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiva, Japan, www.ncc.go.jp/en/epoc/division/immunotherapy/kashiwa/020/020/20170728115215.html
- Guy Schoehn, Electron Microscopy Group, Institute of Structural Biology, Grenoble, France, ibs.fr/research/research-groups/methods-and-electron-microscopy-group/schoehn-team/?lang=fr
- University of Warsaw, Poland
- Nencki Institute of Experimental Biology Polish Academy of Sciences , Warsaw, Poland
Our studies focus on the application of adenoviral dodecahedron (Dd), a noninfectious virus-like particle (VLP) that is endowed with remarkable internalization capacity, which makes it a very attractive nanocarrier for direct intracellular delivery.
Dodecahedron particles self-assemble upon production in baculovirus-insect cell expression systems. We optimized particle production and elaborated an efficient scalable two-step purification procedure, as well as a lyophilization method that enables long-term storage at a broad spectrum of temperatures.
Crystallographic analysis combined with site-directed mutagenesis revealed molecular mechanisms of Dd assembly and the requirements for VLP stability. These findings were the basis for the development of methods of the chemical attachment of therapeutic molecules, such as low-molecular-weight drugs or nucleotides, and the incorporation of peptides into the VLP structure through genetic manipulation.
We used Dd with the aim of improving the therapeutic index of toxic cancer drugs and as a carrier for anticancer agents that do not freely diffuse across plasma membrane, such as synthetic mRNA cap analog – the putative inhibitor of oncogene, the initiation translation factor eIF4E. In an animal model of hepatocellular carcinoma (liver cancer), the simultaneous application of Dd conjugates with a cap structure analog and doxorubicin resulted in the significant inhibition of tumor growth, accompanied by a considerable decrease in the levels of two oncogenes, eIF4E and c-myc.
Moreover, the in vitro analysis of Dd affinity for cellular lipids revealed a direct VLP interaction with phosphatidylserine (PS). The involvement of PS in Dd endocytosis suggests that tumors that abundantly expose PS may be considered a target for the efficient and selective Dd-mediated delivery of anti-cancer agents.
Importantly, in vitro studies demonstrated that Dd-mediated delivery overcomes the action of the P-glycoprotein efflux transporter that is responsible for multidrug resistance, which is one of the main reasons of unsuccessful cancer therapy.
Another application of Dd, typical for VLP, is in vaccine design as a self-adjuvanting delivery platform for foreign antigens. Using our innovative approach, we used Dd to engineer a vaccine that carries both hemagglutinin (HA) and epitopes that derive from highly conserved M1 matrix protein for the induction of neutralizing antibodies and cross-reactive cytotoxic T lymphocytes. Upon the intranasal vaccination of chickens with the Dd-M1-HA candidate vaccine, the long-lasting cellular anti-M1 and humoral anti-HA immune responses were elicited in the absence of an adjuvant. These results suggest that the use of adenoviral virus-like particles as a vaccination platform might eliminate the necessity of the addition of potentially harmful substances.
In conclusion, our studies proved that adenoviral Dd is a promising vector candidate for further therapeutic development.
- The structural basis for the integrity of adenovirus Ad3 dodecahedron. Szolajska E, Burmeister WP, Zochowska M, Nerlo B, Andreev I, Schoehn G, Andrieu JP, Fender P, Naskalska A, Zubieta C, Cusack S, Chroboczek J.PLoS One. 2012. doi: 10.1371/journal.pone.0046075.
- Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma. Zochowska M, Piguet AC, Jemielity J, Kowalska J, Szolajska E, Dufour JF, Chroboczek J. Nanomedicine. 2015. doi: 10.1016/j.nano.2014.07.009.
- Cholesterol and phosphatidylserine are engaged in adenoviral dodecahedron endocytosis. Jedynak M, Worch R, Podsiadła-Białoskórska M, Chroboczek J, Szołajska E. BBA – Biomembranes. 2018. doi: 10.1016/j.bbamem.2018.09.002.
- Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron. Jedynak M, Laurin D, Dolega P, Podsiadla-Bialoskorska M, Szurgot I, Chroboczek J, Szolajska E. 2018. doi: 10.1016/j.nano.2018.05.001
Prizes and Awards
- Antonina Naskalska. Award for the PhD thesis in Competition for Best Master’s and Doctor’s 2011. Hasco-Lek Foundation. Poland.
nuclear localization of hexons from subgroups C and B adenoviruses. Journal of Molecular Biology (2005) 352: 125-138 IF 4,89
- Jan Kowalski
- Virus-like particle vector as a polyvalent platform for intracellular delivery of high-molecular-weight therapeutic substances, method for generating a virus like particle vector and use of a virus-like particle vector and a pharmaceutical composition containing said virus-like particle vector. Szolajska E, Chroboczek J, Naskalska A. 2016. EP2461827.