Extending Janus lectins architecture: Characterization and application to protocells

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being exploited for producing tools with precise topology and specificity. We developed the concept of engineered chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the 0-propeller domain from Ralstonia solanacearum and the 0-trefoil domain from fungus Marasmius oreades, is specific for fucose and a-galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The engineered protein recognizes and binds H1299 human lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.(c) 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Automated summary

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Engineered chimeric lectins have been developed to produce tools with precise topology and specificity. The novel engineered lectin can bind fucose and a-galactose simultaneously and form tissue-like structures through cross-linking of glycosylated giant unilamellar vesicles. This study explores the importance of protein engineering.