Difference between revisions of "Part:BBa K5185017"

 
 
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<partinfo>BBa_K5185017 short</partinfo>
 
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a2-sumo-HNP1 is a fusion protein with three distinct domains: Human Neutrophil Peptide 1 (HNP1 <partinfo>BBa_K5185000</partinfo>), a natural antimicrobial peptide belonging to the &#945;-defensin family that exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses, integrin &#945;2 domain (&#945;2 <partinfo>BBa_K5185004</partinfo>), and the SUMO tag <partinfo> BBa_K4170016</partinfo>, which can improve the solubility, stability, and folding of proteins.
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α2-sumo-HNP1 is a fusion protein with three distinct domains: Human Neutrophil Peptide 1 (HNP1, <partinfo>BBa_K5185000</partinfo>), 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, integrin α2 domain α2 (<partinfo>BBa_K5185004</partinfo>) which attach defensins to collagen-containing or laminin-containing materials, and the SUMO tag 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.
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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.
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<!-- Add more about the biology of this part here
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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 α2 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of collagen or laminin, with properties that facilitate hemostasis and prevents bacterial growth. α2 links defensins to collagen or laminin containing materials, while HNP1 interferes with the normal functionality of bacteria.
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===Usage and Biology===
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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.
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Other than α2, this part collection includes other CBMs such as CBM2, CBM3, CBM5, and VbCBMxx, and also the human integrin domain α1. Other than HNP1, other defensins in this part collection include HNP4, HD5, and HBD3. We synthesized the fusion proteins CBM3-sumo-HNP1 (<partinfo>BBa_K5185010</partinfo>), CBM3-sumo-HNP4 (<partinfo>BBa_K5185011</partinfo>),CBM3-sumo-HD5 (<partinfo>BBa_K5185012</partinfo>), and CBM3-sumo-HBD3 (<partinfo>BBa_K5185013</partinfo>) for materials in the first aid kit composed of cellulose, bestowing them with antimicrobial functions. Other fusion proteins we synthesized include CBM5-sumo-HNP1 (<partinfo>BBa_K5185015</partinfo>) which focuses on more enhanced anti-microbial functions and targets especially severe infections, and α2-sumo-HNP1 (<partinfo>BBa_K5185017</partinfo>) 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.
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Usage and Biology:
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a2-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 surgical sutures. 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.
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===Results===
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After plasmid construction, we successfully expressed and purified α2-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.
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<img style="display: block;
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    width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/sumo-hnp1.png" text-align="center"><div>Figure 1:</div></html>
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(a) SDS-PAGE analysis of the target fusion proteins (binding domains linking SUMO and defensins) expression in E. coli SHuffle T7 α2-sumo-HNP1 has a molecular weight of 36kDa and is successfully expressed.
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<br> (b) Antibacterial assay of α2-SUMO↓HNP1 and CBM5-SUMP↓HNP1 against E. Coli and S. aureus
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After SUMO cleavage, α2-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.
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===Reference===
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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.
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<br> 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
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<br> 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
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<br> 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
  
 
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Latest revision as of 11:45, 2 October 2024


α2-sumo-HNP1

α2-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, integrin α2 domain α2 (BBa_K5185004) which attach defensins to collagen-containing or laminin-containing materials, and the SUMO tag 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 α2 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of collagen or laminin, with properties that facilitate hemostasis and prevents bacterial growth. α2 links defensins to collagen or laminin 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 α2, this part collection includes other CBMs such as CBM2, CBM3, CBM5, and VbCBMxx, and also the human integrin domain α1. 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: a2-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 surgical sutures. 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

After plasmid construction, we successfully expressed and purified α2-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.

Figure 1:
(a) SDS-PAGE analysis of the target fusion proteins (binding domains linking SUMO and defensins) expression in E. coli SHuffle T7 α2-sumo-HNP1 has a molecular weight of 36kDa and is successfully expressed.
(b) Antibacterial assay of α2-SUMO↓HNP1 and CBM5-SUMP↓HNP1 against E. Coli and S. aureus After SUMO cleavage, α2-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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 646
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 646
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 882
    Illegal BamHI site found at 25
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 646
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 646
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 623