The activities of the Bio-Nanosystems Research Laboratory play a fundamental role in researching the organizational and operational principles of self-assembling supramolecular systems built from proteins, with applications in bio- and nanotechnology. Flagella are the bacterial motility organs composed of a nanomotor embedded in the cell membrane and a helical filament rotated as a propeller. The flagellar filaments are made up of thousands of copies of the flagellin protein. Flagellin is capable of self-assembly, and under suitable conditions, nanotubes with the desired size distribution can be built from it. It has been demonstrated that other proteins or peptide motifs can be genetically incorporated into the hypervariable D3 domain of flagellin subunits without disrupting their polymerization ability. One of our main research directions is the application of flagellin protein variants endowed with various functions to create nanostructures with advantageous properties. By incorporating small, specific binding proteins (e.g., single-domain antibodies, binding motifs) into the variable region of flagellin, we create variants capable of recognizing and efficiently binding to a target molecule.
Modified flagellins are used to build nanorods of desired size with high surface binding site density, which can serve as sensing layers for detecting specific target molecules. For environmental analytical applications, flagellin protein has been modified to be capable of binding to pollutant heavy metal ions (e.g., Ni, Co, As) for their detection. By modifying or exchanging the variable domain, we aim to create variants that are selective for certain metal ions or capable of detecting multiple pollutants simultaneously. Flagellin variants have also been produced containing various cell surface receptor binding motifs while retaining their self-assembly ability. Protein coatings formed from these on optical waveguide sensors enable real-time studies of the adhesion properties and behavior of cancer cells.
By incorporating protein segments into the D3 domain, which play a crucial role in the formation of magnetite nanocrystals in magnetic bacteria, nanorods with periodically repeating magnetite binding sites can be created from mutant flagellins. These modified filaments provide a favorable template surface for the formation and growth of magnetic iron oxide crystals, thus enabling the bioinspired environmentally friendly production of magnetic nanotubes. By incorporating the fluorescent GFP protein into the interior of flagellin, tracer units can also be produced. It has also been demonstrated that enzymes capable of flagellin-based polymerization can be created, from which we hope to construct supramolecular multi-enzyme systems that can be beneficially used in biocoversion technologies. Our long-term goal is to build multifunctional filamentous nanostructures using flagellin variants endowed with various properties. The successful research and development activities of the group are reflected in several national patents. The Bio-Nanosystems Research Laboratory was awarded the Millennium Prize by the National Intellectual Property Office in 2014.
RESEARCH GROUP LEADER
Dr. Hajnalka Jankovics
Senior Research Fellow
Contact
Services and Infrastructure
Biosynthesis and sterile sample preparation options
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
Fermentors, laminar flow cabinets, shaking incubators, ultrasonic disruptors (sonicators) for cell disruption, autoclave, ultra-deep freezer.
Ultracentrifuges
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
1.) The Beckman Coulter Optima™ MAX-XP desktop ultracentrifuge is used for separating cell components and polymers, including protein monomers. It comes with 4 different-sized titanium rotors (8 ∙ 6.5 ml; 6 ∙ 3 ml; 8 ∙ 3.5 ml; 12 ∙ 1 ml), and depending on the rotor, it can be operated at speeds of up to 80,000-120,000 rpm (up to 600,000 g). It can be cooled down to 10°C. 2.) The Beckman Coulter Optima MAX-E desktop ultracentrifuge is also used for separating cell components and polymers, including protein monomers. It comes with 4 different-sized titanium rotors (8 ∙ 6.5 ml; 6 ∙ 3 ml; 8 ∙ 3.5 ml; 12 ∙ 1 ml), and depending on the rotor, it can be operated at speeds of up to 80,000-100,000 rpm (up to 500,000 g). It can be cooled down to 10°C. 3.) The Sorvall Discovery 90SE floor-standing ultracentrifuge is used, for instance, for separating cell components and polymers, including protein monomers. It comes with several different-sized titanium rotors (6 ∙ 120 ml; 6 ∙ 50 ml), and depending on the rotor, it can be operated at speeds of up to 16,000-40,000-65,000 rpm (up to 400,000 g). It can be cooled down to 10°C.
PCR instruments
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
1.) Biometra T-Personal PCR - A programmable thermal block suitable for conducting simple polymerase chain reactions. It can be used in the temperature range of +3.0°C to +99.0°C, with a maximum heating rate of 3°C/s and maximum cooling rate of 3°C/s, with 0.1°C accuracy. It accommodates 0.2 and 0.5 ml (24 + 24) sample tubes. 2.) SensoQuest Labcycler gradient PCR - A programmable thermal block suitable for parallel optimization of temperature parameters for polymerase chain reactions. It can be used in the temperature range of -5.0°C to +99.9°C, with a heating rate of 4.2°C/s and a cooling rate of 3.6°C/s. There can be a difference of ±20°C between the two ends of the gradient. It allows for setting up multiple parallel PCR reactions with 0.01°C accuracy. It can accommodate a maximum of 96 0.2 ml tubes.
CD spectroscopy
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
The Jasco J1100 CD spectrometer operates in the wavelength range of 180 to 600 nm, suitable for examining the secondary and tertiary structural elements of proteins, as well as their thermal stability in the near and far UV regions. Equipped with a Peltier cell holder, the J-1100 can be used for CD, LD, and absorbance measurements as a function of wavelength, temperature, and time.
FPLC chromatography systems
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
1.) ÄKTA Start FPLC system - Used for laboratory-scale preparative purification of proteins, mainly for affinity chromatography. It operates at flow rates of 0.5-5 ml/min, equipped with a fixed 280 nm wavelength UV detector and conductivity measurement capability. 2.) ÄKTA Pure 25M FPLC system - Enables rapid preparative purification of proteins, peptides, and nucleic acids based on liquid chromatography. It operates at flow rates of 0.1-25 ml/min, equipped with fixed wavelength UV detectors at 254 nm and 280 nm, and conductivity measurement capability. It supports ion exchange, size exclusion, and various affinity chromatography columns.
Darkfield and fluorescence microscope
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
The Olympus BX50 darkfield, fluorescence-capable microscope features 10x, 40x, and 100x objectives, with two illumination positions and the capability to attach a camera. It is used for monitoring and observing unstained bacterial cells.
Fluorescence and UV spectrophotometers
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
1.) Fluoromax-2 - Equipped with Czerny-Turner spectrometers in both excitation and emission positions, usable in the range of 200-900 nm. 2.) JASCO V-630 spectrophotometer - Dual-beam, 190-900 nm, for quantitative analysis of components of solutions with absorption in the UV-Vis range. 3.) NANO-ND-LITE photometer - Measures at fixed wavelengths in a sample volume of 1-2 microliters, determining nucleic acid concentration and purity at 260 nm using the 260/280 ratio, and pure protein concentration at 280 nm.
Isothermal titration microcalorimeter
Research Institute of Biomolecular and Chemical Engineering | Bio-Nanosystems Laboratory
Research field: Recombinant protein design and synthesis, flagellin-based nanosheets and nanofibers
1.) Fluoromax-2 - Equipped with Czerny-Turner spectrometers in both excitation and emission positions, usable in the range of 200-900 nm. 2.) JASCO V-630 spectrophotometer - Dual-beam, 190-900 nm, for quantitative analysis of components of solutions with absorption in the UV-Vis range. 3.) NANO-ND-LITE photometer - Measures at fixed wavelengths in a sample volume of 1-2 microliters, determining nucleic acid concentration and purity at 260 nm using the 260/280 ratio, and pure protein concentration at 280 nm.
Partners
- Műszaki Fizikai és Anyagtudományi Intézet (MFA), Budapest - Nanobioszenzorika Lendület Kutatócsoport - Dr. Horváth Róbert
- Műszaki Fizikai és Anyagtudományi Intézet (MFA), Budapest- Fotonika Laboratórium - Dr. Petrik Péter
- Soós Ernő Kutató-Fejlesztő Központ, Nagykanizsa - Dr. Galambos Ildikó
- BioSense Institute (Serbia) - Center for sensing technologies - Dr. Vasa Radonić
- TargetEx Biosciences - Dr. Lőrincz Zsolt
Publications
- Farsang R, Kovács N, Szigeti M, Jankovics H, Vonderviszt F, Guttman A (2022) Immobilized exoglycosidase matrix mediated solid phase glycan sequencing. Analytica Chimica Acta 1215:339906.
- Jankovics H, Szekér P, Tóth É, Kakasi B, Lábadi Z, Saftics A, Kalas B, Fried M, Petrik P, Vonderviszt F. (2021) Flagellin-based electrochemical sensing layer for arsenic detection in water. Scienctific Reports 11:3497.
- Jankovics H, Kovacs B, Saftics A, Gerecsei T, Tóth É, Szekacs I, Vonderviszt F, Horvath R (2020) Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers. Scientific Reports 10:22253.
- Labadi Z, Kalas B, Saftics A, Illes L, Jankovics H, Bereczk-Tompa E, Sebestyen A, Toth E, Kakasi B, Moldovan C, Firtat B, Gartner M, Gheorge M, Vonderviszt F, Fried M, Petrik P (2020) Sensing Layer for Ni Detection in Water Created by Immobilization of Bioengineered Flagellar Nanotubes on Gold Surfaces. ACS Biomaterials Science & Engineering 6: 3800-3820.