Abstract:

Mussel foot proteins (Mfps) are a class of proteins secreted by the byssal threads of mussels, exhibiting exceptional underwater adhesion, water resistance, mechanical toughness, and long-term durability. These properties confer significant application potential in biomedical engineering, cosmetics, and medical coating technologies. However, current Mfp production predominantly relies on direct extraction from natural sources, a process that is associated with high costs, low efficiency, and inconsistent yields and quality, thereby limiting scalability and commercial feasibility. To address this limitation, this study aims to develop Escherichia coli as a heterologous expression platform for the efficient biosynthesis of Mfps. Furthermore, by leveraging protein sequence engineering and molecular design strategies, we propose to construct multifunctional fusion proteins integrating robust adhesive capabilities, enhanced antibacterial activity, and excellent biocompatibility, with the goal of developing antimicrobial coatings for medical devices.

To achieve the project's design objectives, we systematically investigated key functional components from three distinct aspects: adhesive proteins, antibacterial peptides, and anti-fouling zwitterionic peptides.

Mfp3 and Mfp5 are mussel foot proteins known for their exceptional adhesive properties in wet environments, primarily due to the presence of abundant 3,4-dihydroxyphenylalanine (DOPA) residues that facilitate strong and versatile interactions with various surfaces. These unique biochemical features make Mfp3 and Mfp5 ideal candidates as adhesive modules in bio-coatings, enabling robust and durable attachment even under challenging conditions, which is crucial for the development of effective and stable biological coatings.

Antimicrobial peptides (AMPs) serve as a critical functional module in bio-coating fusion proteins due to their broad-spectrum and potent antibacterial activities. We selected D51, Melittin, and Tet213 antimicrobial peptides based on their ability to effectively disrupt bacterial membrane integrity and rapidly kill a wide range of drug-resistant bacterial strains. In addition to their strong bactericidal effects, these AMPs also exhibit immunomodulatory and anti-inflammatory properties, which contribute to enhanced biocompatibility. Incorporating these peptides into the coating significantly improves its antimicrobial performance, effectively preventing bacterial invasion and biofilm formation. This ensures the long-term stability and biological safety of the bio-coating, making it highly suitable for diverse biomedical and industrial applications.

We chose the zwitterionic Poly(KE)15 peptide as the antifouling module in the bio-coating fusion protein due to its excellent resistance to nonspecific protein adsorption and microbial adhesion. Zwitterionic peptides possess balanced positive and negative charges that create a strong hydration layer on the coating surface, effectively preventing the attachment of proteins, cells, and biofilm formation. This unique antifouling property helps maintain the coating’s cleanliness and functionality over time, enhancing its durability and biocompatibility in complex biological environments.

Parts list：

In this study, all the functional components (parts) involved in the project were successfully constructed or synthesized, and systematic verification was carried out. The results showed that they possess excellent functional characteristics.

Reference：

[1] Yu, J., Wei, W., Danner, E., Ashley, R. K., Israelachvili, J. N., & Waite, J. H. (2011). Mussel protein adhesion depends on interprotein thiol-mediated redox modulation. Nature chemical biology, 7(9), 588–590. https://doi.org/10.1038/nchembio.630

[2] Zhao, H., & Waite, J. H. (2006). Linking adhesive and structural proteins in the attachment plaque of Mytilus californianus. The Journal of biological chemistry, 281(36), 26150–26158. https://doi.org/10.1074/jbc.M604357200

[3] Hwang, D. S., Zeng, H., Masic, A., Harrington, M. J., Israelachvili, J. N., & Waite, J. H. (2010). Protein- and metal-dependent interactions of a prominent protein in mussel adhesive plaques. The Journal of biological chemistry, 285(33), 25850–25858. https://doi.org/10.1074/jbc.M110.133157

[4] Lee, B. P., Messersmith, P. B., Israelachvili, J. N., & Waite, J. H. (2011). Mussel-Inspired Adhesives and Coatings. Annual review of materials research, 41, 99–132. https://doi.org/10.1146/annurev-matsci-062910-100429

[5] Zhang, L., Cao, Z., Bai, T., Carr, L., Ella-Menye, J. R., Irvin, C., Ratner, B. D., & Jiang, S. (2013). Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nature biotechnology, 31(6), 553–556. https://doi.org/10.1038/nbt.2580

[6] Loose, C., Jensen, K., Rigoutsos, I., & Stephanopoulos, G. (2006). A linguistic model for the rational design of antimicrobial peptides. Nature, 443(7113), 867–869. https://doi.org/10.1038/nature05233

[7] Yanmei Li, Meng Wang, Yuqi Li, Bin Hong, Duo Kang, Yi Ma, Jufang Wang, Two novel antimicrobial peptides against vegetative cells, spores and biofilm of Bacillus cereus, Food Control, Volume 149,2023, 109688,ISSN 0956-7135, https://doi.org/10.1016/j.foodcont.2023.109688.

[8] Raghuraman, H., & Chattopadhyay, A. (2007). Melittin: a membrane-active peptide with diverse functions. Bioscience reports, 27(4-5), 189–223. https://doi.org/10.1007/s10540-006-9030-z

[9] Oršolić N. (2012). Bee venom in cancer therapy. Cancer metastasis reviews, 31(1-2), 173–194. https://doi.org/10.1007/s10555-011-9339-3

[10] Pwa B, et al. Antibacterial peptide-modified collagen nanosheet for infected wound repair - ScienceDirect[J]. Smart Materials in Medicine, 2021.

[11] Zhao G, et al. Effects of antimicrobial peptides on Staphylococcus aureus growth and biofilm formation in vitro following isolation from implant-associated infections. Int J Clin Exp Med. 2015 Jan 15;8(1):1546-51.