Construction of Bifunctional Protein/Peptide Complexfor Sensitive Detection of Transglutaminase 2

Bifunctional protein complexes play essential roles in the biomedical field, particularly in biochemical analysis. However, traditional protein engineering methods (e.g., gene fusion and covalent modification) suffer limitations of complex design, low efficiency, and lack of universality. In this work, we propose a straightforward, efficient, and universal strategy for the preparation of a bifunctional protein complex. Green fluorescent protein (GFP) is engineered using the protein purification tag and coordinate-mediated peptide assembly, facilitating both target recognition and signal reporting. It is further applied to develop a sensitive biosensor for the detection of a target protein. Due to the substantial loading of functional components within the complex, the proposed biosensor demonstrates a simple procedure and high sensitivity. Furthermore, the analysis of clinical samples has been achieved to distinguish breast cancer patients from healthy individuals. Given the abundance of histidine-tagged proteins and the customizable nature of peptides, this work is expected to provide a valuable concept for bifunctional protein engineering in biosensing and broader biomedical applications.