Photocrosslinkable triple helical protein with enhanced higher-order formation for biomaterial applications.

Bacterial collagen, produced via recombinant DNA methods, offers advantages including consistent purity, customizable properties, and reduced allergy potential compared to animal-derived collagen. Its controlled production environment enables tailored features, making it more sustainable, non-pathogenic, and compatible with diverse applications in medicine, cosmetics, and other industries. Research has focused on the engineering of collagen-like proteins to improve their structure and function. The study explores the impact of introducing tyrosine, an amino acid known for its role in fibril formation across diverse proteins, into a newly designed bacterial collagen-like protein (Scl2), specifically examining its effect on self-assembly and fibril formation. Biophysical analyses reveal that the introduction of tyrosine residues didn't compromise the protein's structural stability but rather promoted self-assembly, resulting in the creation of nanofibrils-a phenomenon absent in the native Scl2 protein. Additionally, stable hydrogels are formed when the engineered protein undergoes di-tyrosine crosslinking under light exposure. The hydrogels, shown to support cell viability, also facilitate accelerated wound healing in mouse fibroblast (NIH/3T3) cells. These outcomes demonstrate that the targeted inclusion of functional residues in collagen-like proteins enhances fibril formation and facilitates the generation of robust hydrogels using riboflavin chemistry, presenting promising paths for research in tissue engineering and regenerative medicine.

Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling.

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein-protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-l-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-l-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.