Renaudet, O. | Search bioRxiv

bioRxiv2023

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free protein synthesis as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with BLI analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

synthetic biology↗

bioRxiv2019

Development Of C-Type Lectin Oriented Surfaces For High Avidity Glycoconjugates: Towards Mimicking Multivalent Interactions On The Cell Surface

Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, {micro}M). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides a microscopic overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers that are organized in nanoclusters on the cell membrane. For this purpose, covalent biospecific capture of DC-SIGN via StreptagII /StrepTactin interaction offers the preservation of tetrameric DC-SIGN and the accessibility/functionality of all active sites. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3.D illustrates the tightest binding partner in this set for a DC-SIGN surface (Kd= 18 nM). Moreover, the selectivity at monovalent scale of glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible to avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immunomodulators.

biochemistry↗