Part:BBa_K5185015
CBM5-sumo-HNP1
CBM5-sumo-HNP1 is a fusion protein combining three distinct domains: Human Neutrophil Peptide 1 (HNP1BBa_K5185000 ), a natural antimicrobial peptide belonging to the alpha defensin family that exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses, Carbonhydrate-Binding molecule 5(CBM5 BBa_K5185002), which enhances the targeting of chitosan-containing materials for modification or degradation, and the SUMO tag which can improve the solubility, stability, and folding of proteins. The universality of the combined function of the binding domain and defensins of this part is assessed. The fusion protein allowing for a more versatile collection of antibacterial dressings and enhanced potential of our first aid kit to address more complex situations.
CBM5-sumo-HNP1 is a fusion protein with three distinct domains: Human Neutrophil Peptide 1 (HNP1, BBa_K5185000), a natural antimicrobial peptide belonging to the α-defensin family that exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses, Carbonhydrate-Binding molecule 5 (CBM5 BBa_K5185002)that allows the defensin to attach to chitosan-containing materials of the first aid kit, and the SUMO tag (BBa_K4170016) which improves the solubility, stability, and folding of proteins. This part is part of a collection where the universality of the combined function of the binding domain and defensins is assessed, allowing for a more versatile collection of antibacterial dressings and enhancing the potential of our first aid kit to address more complex situations.
Our project aims to endow first-aid wound dressings with enhanced antimicrobial functions and a wider and more complex application. By fusing the binding domain CBM5 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of chitosan, with properties that facilitate hemostasis and prevents bacterial growth. CBM5 allows the HNP to attach to chitosan-containing materials, while HNP1 interferes with the normal functionality of bacteria.
This is part of a part collection of SUMO linking a binding domain to defensin, which allows defensins to be attached to carbohydrates such as cellulose, chitosan, and collagen. When applied with a SUMO Protease, this fusion protein may effectively release the defensin into the site of injury and therefore achieve the desired antimicrobial effects, acting as a reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance.
Other than CBM5, this part collection includes other CBMs such as CBM2, CBM3, and VbCBMxx, and also human integrin domains such as α1 and α2. Other than HNP1, other defensins in this part collection include HNP4, HD5, and HBD3. We synthesized the fusion proteins CBM3-sumo-HNP1 (BBa_K5185010), CBM3-sumo-HNP4 (BBa_K5185011),CBM3-sumo-HD5 (BBa_K5185012), and CBM3-sumo-HBD3 (BBa_K5185013) for materials in the first aid kit composed of cellulose, bestowing them with antimicrobial functions. Other fusion proteins we synthesized include CBM5-sumo-HNP1 (BBa_K5185015) which focuses on more enhanced anti-microbial functions and targets especially severe infections, and α2-sumo-HNP1 (BBa_K5185017) which with the use of collagen enables better wound healing, targeting wounds that prioritize wound recovery. Recognizing this part collection of fusion proteins as effective in treating wounds and achieving antimicrobial needs, we believe the HNPs could each be linked with different wound-dressing materials that would provide an array of approaches and solutions to suit the varied needs of different wounds and circumstances with limited medical resources, such as battlefields and disaster zones.
Usage and Biology: CBM5-sumo-HNP1 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, making it a suitable protein for antimicrobial coatings on wound dressings, biodegradable antibacterial materials, or biofilm disruption on cellulose-based surfaces. HNP1 disrupts microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and CBM5 increases the catalytic efficiency by creating a better fit between the enzyme and substrate surfaces.
Results: After plasmid construction, we successfully expressed and purified CBM5-sumo-HNP1. We then performed SUMO cleavage and antibacterial tests. Both CBM5-SUMO↓HNP1 and α2-SUMO↓HNP1 demonstrated inhibitory effects on Escherichia coli and Staphylococcus aureus. Therefore, the function of our design (Binding domain-SUMO-Defensins) is verified and is scalable.
(b) Antibacterial assay of α2-SUMO↓HNP1 and CBM5-SUMP↓HNP1 against E. Coli and S. aureus After SUMO cleavage, CBM5-SUMO↓HNP1 demonstrated inhibitory effects on both Escherichia coli and Staphylococcus aureus in zone of inhibition tests. Antimicrobial function of HNP1 after SUMO tag cleaving by the Ulp1 enzyme is verified.
Reference
Armenta, S., Moreno-Mendieta, S., Sánchez-Cuapio, Z., Sánchez, S., & Rodríguez-Sanoja, R. (2017). Advances in molecular engineering of carbohydrate-binding modules. Proteins, 85(9), 1602–1617.
Han, Y., Gao, P., Yu, W., & Lu, X. (2017). Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module. Biotechnology letters, 39(12), 1895–1901. https://doi.org/10.1007/s10529-017-2406-2
Heino, J., Siljamäki, E. (2023). Integrins α1β1 and α2β1: The Generalist Collagen Receptors. In: Gullberg, D., Eble, J.A. (eds) Integrins in Health and Disease. Biology of Extracellular Matrix, vol 13. Springer, Cham. https://doi.org/10.1007/978-3-031-23781-2_1
Mei, X., Tao, W., Sun, H., Liu, G., Chen, G., Zhang, Y., Xue, C., & Chang, Y. (2024). Characterization and structural identification of a novel alginate-specific carbohydrate-binding module (CBM): The founding member of a new CBM family. International journal of biological macromolecules, 277(Pt 3), 134221. https://doi.org/10.1016/j.ijbiomac.2024.134221
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 61
Illegal NgoMIV site found at 126 - 1000COMPATIBLE WITH RFC[1000]
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