Construction of a Xylanase A Variant Capable of Polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created

Nanobody-Displaying Flagellar Nanotubes

In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology

Proteolytic sensitivity of FliC(XynA) in the polymeric and monomeric form.

<p>(A) Copolymers of flagellin and FliC(XynA) obtained by 0.6 M AS at 1∶1 mixing ratio (w/w) were incubated with trypsin at a 30∶1 (w/w) ratio. (B) Proteolysis of monomeric FliC(XynA) at a protein to protease ratio of 300∶1 (w/w). At the indicated time points, portions were removed from the reaction mixtures, mixed with electrophoresis sample buffer and boiled for 3 min. Experiments were done in 20 mM Tris–HCl, 150 mM NaCl (pH 7.8) at room temperature at a 1 mg/ml protein concentration.</p

Catalytic activity of recombinant xylanase A and the FliC(XynA) fusion protein in the monomeric and polymeric forms.

<p>Measurements were done at 1.2 µM enzyme (catalytic unit) concentration in 50 mM phosphate buffer (pH 6.0) at 37°C. The amount of FliC(XynA) subunits incorporated into the copolymers was estimated by densitometric analysis of SDS-PA gels of filament samples.</p

Design of the FliC(XynA) fusion construct.

<p>The hypervariable D3 domain of Salmonella flagellin (residues 190–284) was replaced by xylanase A from <i>B. subtilis</i>. The C_α backbone trace of flagellin and XynA is rainbow-colored from blue to red representing the sequence from the NH₂- to COOH terminus.</p

Products of the polymerization of FliC(XynA) subunits.

<p>Dark-field light micrographs of (A) FliC(XynA) filaments (0.6 M AS) and (B) copolymers of flagellin and FliC(XynA) at 1∶1 (w∶w) mixing ratio (0.4 M AS). Polymerization experiments were done in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl at 1 to 1.5 mg/ml protein concentration, and 4 M AS was added to a final concentration indicated in parentheses to initiate filament formation. (C) SDS/PAGE analysis of copolymer samples polymerized at FliC(XynA) to flagellin ratios of 1∶1 and 1∶2. After AS-induced (0.6 M) polymerization, samples were centrifuged and the pellet was dissolved in 20 mM Tris-HCl (pH 7.8) buffer containing 150 mM NaCl. The molecular masses of flagellin and FliC(XynA) are 51.5 kDa and 64 kDa, respectively. Band intensities determined by densitometry were {49.3 (1∶1); 50.7 (1∶2)} and {49.6 (1∶1) and 34.2 (1∶2)} for FliC and FliC(XynA), respectively. (D) Copolymers observed by atomic force microscopy.</p