Part:BBa_K5185018
VbCBMxx-sumo-HNP1
VbCBMxx-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, VbCBMxx domain (BBa_K5185008), 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 VbCBMxx with the defensin HNP1, we can bestow sodium-alginate-containing items of the first aid kit such as hydrogels with properties that facilitate hemostasis and prevents bacterial growth. VbCBMxx helps the HNP to bind to sodium-alginate-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 VbCMBxx, this part collection includes other CBMs such as CBM2, CBM3, and CBM5, 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
VbCBMxx-sumo-HNP1 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, making it a suitable protein for binding to alginate and for antimicrobial coatings on wound dressings or biodegradable antibacterial materials. HNP1 disrupts microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and α2 integrin mediates cell adhesion to collagen and laminin in the extracellular matrix by binding to collagen types I, III, and IV, as well as laminin.
Results
The lanes from left to right are Marker, WT whole cell, CBM5-SUMO-HNP1 (20.3 kDa) cell lysate supernatant, CBM5-SUMO-HNP1 inclusion body, α2-SUMO-HNP1 cell lysate supernatant (38.0 kDa), α2-SUMO-HNP1 inclusion body, CBMxx-SUMO-HNP1 (39.1 kDa) cell lysate supernatant, and CBMxx-SUMO-HNP1 inclusion body.
The results of the SDS-PAGE analysis of the target fusion proteins (binding domains linking SUMO and defensins) expressed in E. coli SHuffle T7 is shown. VbCBMxx was not expressed due to bacterial contamination during the scale-up culture, and time constraints prevented us from repeating the experiment.
Reference
Mei, X., Tao, W., Sun, H., Liu, G., Chen, G., Zhang, Y., ... & 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, 134221.
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