Nanobody-Displaying Flagellar Nanotubes

Abstract

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.

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

“…ing Information, Figure S3 A). This is at least an approximately 10 000-fold impairment compared to the binding constant reported for the wild-type nanobody (1; K d = 1.1 nm for sfGFP [17] and 0.6 nm for GFP [13] ). Unexpectedly,the NPYcaged photobody (5)w as less impaired (approximately 420fold), with a K d of 0.46 AE 0.02 mm (Supporting Information, Figure S3 B).…”

“…[19] Prior to irradiation of the presenting cells,nosfGFP binding could be detected for concentrations up to about 10 mm,consistent with the results from our MST assay.Wethen added sfGFP to cells that had been irradiated for 45 s( l = 365 nm) and could determine a K d of 0.90 AE 0.03 nm ( Figure 3E), nicely fitting with the positive control and consistent with the previously reported binding constant of the wild-type anti-GFPenhancer nanobody. [13,17] Together, these results demonstrated that the photodeprotected photobody regains its full antigen binding affinity.…”

A mesophilic cysteine-less split intein for protein trans -splicing applications under oxidizing conditions

“…We have also developed a high-affinity version of the split CL intein by fusing a nanobody-epitope pair to the N-and C-terminal intein fragments. Importantly, this proximity-conferring dimerization system can be encoded fully recombinantly (68), in contrast to previous semisynthetic interaction pairs (57,69), and exploits the low nanomolar affinity of the anti-GFP nanobody/eGFP complex (49,50). Specific fluorescent labeling of membrane receptors on the surface of mammalian cells was possible with the labeled CysTagintein part added to the growth medium in concentrations as low as 10 nM.…”

“…4C). The efficient binding of the labeled nanobody at such a low concentration close to its K d value (around 1 nM) (49,50) indicated that its binding capacity was completely preserved.…”

Bacterial Flagellum versus Carbon Nanotube: A Review Article on the Potential of Bacterial Flagellum as a Sustainable and Green Substance for the Synthesis of Nanotubes

“…The bacterial flagellum is a locomotive organelle that includes a membrane-embedded nanomotor that rotates long helical filaments. Flagellar filaments are natural protein nanotubes with an outer diameter of 23 nm, which can raise to 20 μm [84]. This indicated that flagellum has extraordinary mechanical properties as it is extremely rigid and has an elastic modulus estimated at The bacterial flagellum is a locomotive organelle that includes a membrane-embedded nanomotor that rotates long helical filaments.…”