Difference between revisions of "Part:BBa K5185011"
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| − | width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/defensins.jpg" text-align="center"><div>Figure 1: SDS-PAGE analysis of the CBM3-SUMO-Defensins expression in E. coli SHuffle T7 | + | width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/defensins.jpg" text-align="center"><div>Figure 1: SDS-PAGE analysis of the CBM3-SUMO-Defensins expression in E. coli SHuffle T7</div></html> |
| + | The molecular weight of the CBM3-SUMO-Defensins is 33.8kDa，34.0 kDa, 33.9kDa，35.5kDa. | ||
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| − | width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/hbd3/sumo.png" text-align="center"><div>Figure 2 | + | width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/hbd3/sumo.png" text-align="center"><div>Figure 2</div></html> |
| − | (a) Verification of SUMO cleavage of CBM3-SUMO-Defensins. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1.(b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus | + | (a) Verification of SUMO cleavage of CBM3-SUMO-Defensins. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1. |
| + | <br> (b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus | ||
4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1. The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 3a), indicating successful removal of ~4 kDa defensins. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 2b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach. | 4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1. The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 3a), indicating successful removal of ~4 kDa defensins. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 2b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach. | ||
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Verification of the antibacterial activity of CBM3-SUMO↓Defensins. | Verification of the antibacterial activity of CBM3-SUMO↓Defensins. | ||
(a) The effect of the four CBM3-SUMO↓Defensins on the growth of Staphylococcus aureus within 24 hours. | (a) The effect of the four CBM3-SUMO↓Defensins on the growth of Staphylococcus aureus within 24 hours. | ||
| − | (b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours. | + | <br> (b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours. |
| − | Antimicrobial assay of HNP4 showed that the HNP4 successfully inhibited the growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag. | + | <br> Antimicrobial assay of HNP4 showed that the HNP4 successfully inhibited the growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag. |
It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 3b). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 3a). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC. | It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 3b). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 3a). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC. | ||
Revision as of 06:32, 2 October 2024
CBM3-sumo-HNP4
CBM3-sumo-HNP4 is a fusion protein with three distinct domains: Human Neutrophil Peptide 4 (HNP4, BBa_K5185001), a member of the α-defensin family of antimicrobial peptides produced by neutrophils, Carbonhydrate-Binding molecule 3(CBM3, BBa_K4011000) that enhances the ability of enzymes to target and degrade cellulose by attaching to the cellulose surface, 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 CBM3 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of cellulose, with properties that facilitate hemostasis and prevents bacterial growth. CBM3 activates hemostatic pathways in the human body, 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 CBM3, this part collection includes other CBMs such as CBM2, CBM5, 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
CBM3-sumo-HNP4 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, which makes it suitable for antimicrobial coatings on wound dressings, biodegradable antibacterial materials, or biofilm disruption on cellulose-based surfaces. HNP4 is able to disrupts microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and CBM3 has applications in biotechnological processes, including biomass conversion, biofuel production, and the recycling of plant materials.
CBM3-sumo-HNP4 is a fusion protein with three distinct domains: Human Neutrophil Peptide 4 (HNP4, BBa_K5185001), a member of the α-defensin family of antimicrobial peptides produced by neutrophils, Carbonhydrate-Binding molecule 3(CBM3, BBa_K4011000) that enhances the ability of enzymes to target and degrade cellulose by attaching to the cellulose surface, 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 CBM3 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of cellulose, with properties that facilitate hemostasis and prevents bacterial growth. CBM3 activates hemostatic pathways in the human body, 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 CBM3, this part collection includes other CBMs such as CBM2, CBM5, 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
CBM3-sumo-HNP4 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, which makes it suitable for antimicrobial coatings on wound dressings, biodegradable antibacterial materials, or biofilm disruption on cellulose-based surfaces. HNP4 is able to disrupts microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and CBM3 has applications in biotechnological processes, including biomass conversion, biofuel production, and the recycling of plant materials.
Results
The IPTG-induced expression of CBM3-SUMO-defensins was validated in the same manner as the defensins. SDS-PAGE analysis revealed the presence of the correct bands (Fig. 1). Subsequently, we scaled up the fermentation to 400 mL. Once the optical density (OD) reached 0.6–0.8, we induced expression with 0.1 mM IPTG at 20°C for 12 hours. Afterward, we lysed the cells and purified the target protein from the cell lysate using a Ni-NTA affinity chromatography column.
(b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus
4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1. The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 3a), indicating successful removal of ~4 kDa defensins. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 2b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach.
We utilized the microdilution method to determine the MIC values of four types of CBM3-SUMO-Defensins. For specific details, please refer to our measurement section. Initially, we examined the 24-hour growth curves of Staphylococcus aureus with the addition of CBM3-SUMO↓Defensins. Within the 0–8 hour range, all four types of CBM3-SUMO↓Defensins exhibited antimicrobial activity (Fig. 2a). We selected the 8-hour time point to define the MIC values against Staphylococcus aureus. At this point, the MIC50 values for CBM3-SUMO↓HNP1, CBM3-SUMO↓HNP4, CBM3-SUMO↓HD5, and CBM3-SUMO↓HBD3 were 0.74 μM, 0.368 μM, 1.475 μM, and 1.001 μM, respectively. Additionally, the MIC90 values for CBM3-SUMO↓HNP4/HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported previously for the four defensins (Wei et al., 2009).
(b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours.
Antimicrobial assay of HNP4 showed that the HNP4 successfully inhibited the growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag.
It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 3b). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 3a). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC.
Reference
Fu, J., Zong, X., Jin, M., Min, J., Wang, F., & Wang, Y. (2023). Mechanisms and regulation of defensins in host defense. Signal Transduction and Targeted Therapy, 8(1), 300.
Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., ... & Lu, W. (2009). Through the looking glass, mechanistic insights from enantiomeric human defensins. Journal of Biological Chemistry, 284(42), 29180-29192.