Silk fibroin, a natural protein derived from Bombyx mori silkworms, has long been recognized for its exceptional biocompatibility, mechanical strength, and tunable degradation profile. These properties have led to widespread use in tissue engineering, wound healing, and controlled drug delivery systems. However, the functional versatility of silk fibroin can be significantly enhanced through site-specific chemical modification. While conventional conjugation strategies rely on non-selective reactions with reactive amino acid side chains such as lysine or cysteine, this study introduces a chemoenzymatic approach that exploits the specificity of O-GalNAc-transferases to install azide-functionalized glycans directly onto serine residues within the fibroin backbone.
The method utilizes azido-modified UDP-GalNAc as a nucleotide sugar donor and recombinant O-GalNAc-transferases (GalNAc-T1, T10, and T13) to catalyze the transfer of GalNAc to hydroxyl groups of serine and threonine residues. Among these enzymes, GalNAc-T1 and T13 demonstrated efficient glycosylation activity on fibroin, as confirmed by fluorescence detection following ligation with DBCO-Sulfo-Cy5. The resulting conjugates showed distinct fluorescent smears on SDS-PAGE gels corresponding to high molecular weight fibroin, indicating successful incorporation of the azide tag. In contrast, GalNAc-T10 failed to modify fibroin due to its dependence on pre-existing O-glycans—confirming the specificity of the enzymatic reaction and serving as a critical control.
To evaluate the practical utility of this functionalization, a scalable reaction was performed using 15 mg of fibroin. After enzymatic glycosylation and subsequent click conjugation with DBCO-Sulfo-Cy5, the modified protein was purified using a 10 kDa cutoff centrifugal filter to remove excess reagents. A portion of the sample was analyzed via agarose gel electrophoresis, revealing strong fluorescence at the expected migration position of intact fibroin, confirming site-specific labeling. The remainder was concentrated and induced to form a hydrogel by incubation at 37 °C. Within one hour, a solid, self-assembled hydrogel formed, demonstrating that the glycosylation process did not interfere with the protein’s ability to undergo hierarchical self-assembly.
Further purification via extraction with 50% ethanol and 0.Integrin αV Antibody In Vivo 75 M NaCl effectively removed unreacted DBCO-Sulfo-Cy5, leaving behind a stable, fluorescent hydrogel that retained its color and structural integrity.Anti-FMC63 scFv Antibody manufacturer This result underscores the covalent nature of the conjugation and highlights the potential for creating bioactive silk-based materials with built-in signaling or tracking capabilities.PMID:35055362 The strategy is not limited to fluorophores; any cycloalkyne-bearing molecule—including drugs, peptides, or biomolecules—can be attached using this modular platform.
In summary, this work establishes a highly selective, enzyme-driven method for modifying silk fibroin with azide tags, enabling precise and efficient bioconjugation through click chemistry. By targeting abundant serine residues via O-GalNAc-transferases, it overcomes the limitations of non-specific modifications while preserving the material’s intrinsic physical and biological functions. This innovation paves the way for next-generation functional silk biomaterials in advanced biomedical applications.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
