Bio-Nanosystems Laboratory

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Biotemplated synthesis of magnetic nanofibers

With the aim of creating one-dimensional magnetic nanostructures, genetically engineered flagellar filaments were produced to display iron-binding sites, and the mutant filaments were used as templates for the nucleation of the magnetic iron oxide magnetite (Fe3O4). In a previous study we used a similar approach to attach pre-synthesized magnetite nanoparticles to genetically engineered flagella (Bereczk-Tompa et al., 2016). All studied mutant protein filaments were demonstrated to be efficient as templates for the synthesis of one-dimensional magnetic nanostuctures under ambient conditions. Even though we used two fundamentally different approaches for the synthesis of magnetic nanofibers – the templated nucleation of magnetite from solution vs. the attachment of pre-existing magnetite nanoparticles to the protein filaments – the final products of both types of experiments were similar, with randomly oriented magnetite nanoparticles partially covering the filamentous biological templates. In an external magnetic field, the viscosity of a suspension of the produced magnetic filaments showed a twofold increase relative to the control sample. The results of magnetic susceptibility measurements were also consistent with the magnetic nanoparticles occurring in linear structures.


Biomimetic coatings from genetically engineered flagellin variants

Our previous work showed that wild-type flagellin forms oriented and dense monolayers on hydrophobic surfaces, where its variable D3 domain is exposed to the solution. we used the flagellin as a carrier molecule to integrate cell-adhesive peptide sequences, which can influence the adhesion of mammalian cells. The D3 domain of the flagellin was replaced with one or more RGD (Arg-Gly-Asp) motifs by employing various linker pairs. The selected linkers can provide different accessibility and flexibility for the cells. The protein adsorption and cell adhesion were monitored with label-free optical biosensors and parallel microscopic investigations. Our experiments proved that based on genetically engineered flagellins surface coatings with tunable cell adhesivity can be fabricated in a straightforward manner. Moreover, the wild-type and genetically engineered variants can be easily produced by bacteria, making the suggested methodologies interesting for practical applications.

Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. These flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell adhesion regulating biomimetic coatings.


Substrate recognition by the flagellar export machinery

Export of external flagellar proteins requires a signal located within their N-terminal disordered part, however, these regions do not share any significant sequence similarity suggesting that the secondary/tertiary structure might be important for recognition by the export gate. NMR experiments were performed to reveal the conformational properties of the flagellin signal sequence in vitro. Our observations raise the possibility that the signal sequence may partially undergo amphipathic helical ordering upon interaction with the recognition unit of the flagellar export machinery in a similar way as revealed for protein import into intracellular eukaryotic organelles mediated by targeting signals of high diversity.

Our recent results have revealed that various portions of the signal region (SR) can be removed without drastically decreasing export efficiency if at least a segment of 8-10 amino acids remains intact. Although the C-terminal part of SR is very highly conserved, it does not play a dominant role in directing the export process. Our observations suggest that a spacer segment of at least 15 amino acids with a nearly arbitrary sequence is required for efficient export.



Design and production of immobilizable recombinant enzymes suitable for partial fragmentation of glycans
The structural variability of protein-bound carbohydrates (also called glycans) depends on the expression level and activity of the enzymes that synthesize them. The structure of glycans can change under pathological conditions, so these molecules can be used as biomarkers in the early diagnosis of certain diseases. Glycans cleaved from carrier protein and fragmented into smaller subunits can be analysed by capillary electrophoresis coupled mass spectrometry (CE-MS).
Cleavage and specific, controlled fragmentation can be accomplished with appropriate enzymes. Using genetic engineering, our research group is able to create such bacterial strains that are capable of producing large quantities of active enzymes. In addition, it is possible to attach the proteins to a solid support, so that the enzymes can be used several times.

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