Difference between revisions of "Part:BBa K5185002"
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Carbohydrate-binding modules (CBMs) are modules with specificity toward different types of carbohydrates when assisting in carbohydrate degradation. Other than CBM5, this part collection includes other CBMs such as CBM2, CBM3, VbCBMxx, and human integrin domains such as α1 and α2. | Carbohydrate-binding modules (CBMs) are modules with specificity toward different types of carbohydrates when assisting in carbohydrate degradation. Other than CBM5, this part collection includes other CBMs such as CBM2, CBM3, VbCBMxx, and human integrin domains such as α1 and α2. | ||
| − | This is part of a part collection aimed to provide first aid wound dressings with enhanced antimicrobial functions and a wider and more complex application, where we characterize bacterial cellulose modification methods and constructs using CBMs as a binding domain, linking HNPs such as HNP1 and HNP4 to carbohydrates such as cellulose and chitosan. By fusing CBM5 to functional proteins, chitosan-containing materials can be effective targets for modification or degradation. The part collection includes: | + | This is part of a part collection aimed to provide first aid wound dressings with enhanced antimicrobial functions and a wider and more complex application, where we characterize bacterial cellulose modification methods and constructs using CBMs as a binding domain, linking HNPs such as HNP1 and HNP4 to carbohydrates such as cellulose and chitosan. By fusing CBM5 to functional proteins, chitosan-containing materials can be effective targets for modification or degradation. The part collection includes: Carbohydrate binding modules CBM2 (<partinfo>BBa_K4011001 </partinfo>) and CBM3 (<partinfo>BBa_K4011000</partinfo>) which bind to cellulose, CBM5 (<partinfo>BBa_K5185002</partinfo>) which binds to chitosan, and VbCBMxx (<partinfo>BBa_K5185008</partinfo>) which binds to sodium alginate. Human integrins α1 domain (<partinfo>BBa_K5185003 </partinfo>) and α2 domain (<partinfo>BBa_K5185004</partinfo>), linking functional proteins to collagen. This part collection can help to achieve modification of cellulose membranes using different modification/functionalization proteins. |
Because chitosan already possesses stand-alone antimicrobial properties, it could be made into a wound dressing with enhanced antimicrobial strength to combat especially severe instances of infection. | Because chitosan already possesses stand-alone antimicrobial properties, it could be made into a wound dressing with enhanced antimicrobial strength to combat especially severe instances of infection. | ||
| − | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
In nature, CBM5 is expressed as a domain within chitinases and other glycosidic hydrolases that degrade chitin, a structural polysaccharide found in fungal cell walls and arthropod exoskeletons. The binding of CBM5 to chitin increases catalytic efficiency by creating a better fit between the enzyme and substrate surfaces. The structure of CBM5 is documented in the Protein Data Bank (accession: 1ED7). | In nature, CBM5 is expressed as a domain within chitinases and other glycosidic hydrolases that degrade chitin, a structural polysaccharide found in fungal cell walls and arthropod exoskeletons. The binding of CBM5 to chitin increases catalytic efficiency by creating a better fit between the enzyme and substrate surfaces. The structure of CBM5 is documented in the Protein Data Bank (accession: 1ED7). | ||
CBM5 is applied in biotechnological processes such as biocontrol of fungal pathogens, processing of chitinous waste, and production of chitooligosaccharides with biomedical significance. | CBM5 is applied in biotechnological processes such as biocontrol of fungal pathogens, processing of chitinous waste, and production of chitooligosaccharides with biomedical significance. | ||
| + | |||
| + | ===Source=== | ||
| + | CBM5 is derived from organisms that produce chitosan-degrading enzymes, such as certain bacteria and fungi. | ||
| + | |||
| + | ===Result=== | ||
| + | |||
| + | We obtained a fusion protein composed of a binding domain protein linked to a fluorescent protein, naming it CBM5-fwYellow. | ||
| + | Figure A shows the results of the SDS-PAGE analysis of the target fusion proteins (binding domains linked to chromoproteins). The CBM5-fwYellow fusion protein was expressed in E. coli BL21 (DE3). CBM5-fwYellow has a molecular weight of 32.1 kDa and is successfully expressed. | ||
| + | The color of CBM5-fwYellow suspended in 20mM Tris-HCl under bluelight is shown in Figure B, expressing the correct color yellow. | ||
| + | CBM5's ability to bind with chitosan is assessed, as shown in Figure C. The results indicate that CBM5 can successfully bind to chitosan material, as shown by the comparative brightness of the yellow color under bluelight. | ||
| + | <html> | ||
| + | <img style="display: block; | ||
| + | width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/binding-domain-sodium-alginate.png" text-align="center"><div>Figure 1: </div></html> | ||
| + | <br>(a) SDS-PAGE identification of BDC shows the results of SDS-PAGE analysis of the target fusion proteins(BDC). All fusion proteins were expressed in E. coli BL21 (DE3), except for CBMxx-gfasPurple, which was expressed in the SHuffle T7 strain due to its low solubility in BL21 (DE3). α1-mRFP, α2-eforRed, CBM2-mTurquoise, CBM3-sfGFP, CBM5-fwYellow, and CBMxx-gfasPurple each have molecular weights of 49.2 kDa, 49.1 kDa, 40 kDa, 46kDa, 32.l kDa, and 49.5 kDa, respectively. | ||
| + | <br>(b)Yellow-light detection of BDC shows the color of each fusion protein suspended in 20 mM Tris HCl under bluelight. | ||
| + | <br>(c) Binding efficiencies of cellulose binding domains CBM2 and CBM3 toward cellulose gauze; binding efficiencies of collagen binding domains α1 and α2, chitosan binding domain CBM5, and alginate binding domain VbCBMxx toward respective hydrogel materials. | ||
| + | |||
| + | ===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. | ||
| + | <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 | ||
| + | <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 | ||
| + | <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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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 12:06, 2 October 2024
Carbohydrate -binding modules 5（CBM5）
Carbohydrate-Binding Module 5 (CBM5) in particular is a protein binding domain found in carbohydrate-active enzymes, particularly those involved in the degradation of crystalline chitin. CBM5 enhances the ability of related enzymes to bind to their polysaccharide substrates such as chitosan, improving catalytic efficiency.
Carbohydrate-binding modules (CBMs) are modules with specificity toward different types of carbohydrates when assisting in carbohydrate degradation. Other than CBM5, this part collection includes other CBMs such as CBM2, CBM3, VbCBMxx, and human integrin domains such as α1 and α2.
This is part of a part collection aimed to provide first aid wound dressings with enhanced antimicrobial functions and a wider and more complex application, where we characterize bacterial cellulose modification methods and constructs using CBMs as a binding domain, linking HNPs such as HNP1 and HNP4 to carbohydrates such as cellulose and chitosan. By fusing CBM5 to functional proteins, chitosan-containing materials can be effective targets for modification or degradation. The part collection includes: Carbohydrate binding modules CBM2 (BBa_K4011001) and CBM3 (BBa_K4011000) which bind to cellulose, CBM5 (BBa_K5185002) which binds to chitosan, and VbCBMxx (BBa_K5185008) which binds to sodium alginate. Human integrins α1 domain (BBa_K5185003) and α2 domain (BBa_K5185004), linking functional proteins to collagen. This part collection can help to achieve modification of cellulose membranes using different modification/functionalization proteins.
Because chitosan already possesses stand-alone antimicrobial properties, it could be made into a wound dressing with enhanced antimicrobial strength to combat especially severe instances of infection.
Usage and Biology
In nature, CBM5 is expressed as a domain within chitinases and other glycosidic hydrolases that degrade chitin, a structural polysaccharide found in fungal cell walls and arthropod exoskeletons. The binding of CBM5 to chitin increases catalytic efficiency by creating a better fit between the enzyme and substrate surfaces. The structure of CBM5 is documented in the Protein Data Bank (accession: 1ED7). CBM5 is applied in biotechnological processes such as biocontrol of fungal pathogens, processing of chitinous waste, and production of chitooligosaccharides with biomedical significance.
Source
CBM5 is derived from organisms that produce chitosan-degrading enzymes, such as certain bacteria and fungi.
Result
We obtained a fusion protein composed of a binding domain protein linked to a fluorescent protein, naming it CBM5-fwYellow.
Figure A shows the results of the SDS-PAGE analysis of the target fusion proteins (binding domains linked to chromoproteins). The CBM5-fwYellow fusion protein was expressed in E. coli BL21 (DE3). CBM5-fwYellow has a molecular weight of 32.1 kDa and is successfully expressed.
The color of CBM5-fwYellow suspended in 20mM Tris-HCl under bluelight is shown in Figure B, expressing the correct color yellow.
CBM5's ability to bind with chitosan is assessed, as shown in Figure C. The results indicate that CBM5 can successfully bind to chitosan material, as shown by the comparative brightness of the yellow color under bluelight.
(a) SDS-PAGE identification of BDC shows the results of SDS-PAGE analysis of the target fusion proteins(BDC). All fusion proteins were expressed in E. coli BL21 (DE3), except for CBMxx-gfasPurple, which was expressed in the SHuffle T7 strain due to its low solubility in BL21 (DE3). α1-mRFP, α2-eforRed, CBM2-mTurquoise, CBM3-sfGFP, CBM5-fwYellow, and CBMxx-gfasPurple each have molecular weights of 49.2 kDa, 49.1 kDa, 40 kDa, 46kDa, 32.l kDa, and 49.5 kDa, respectively.
(b)Yellow-light detection of BDC shows the color of each fusion protein suspended in 20 mM Tris HCl under bluelight.
(c) Binding efficiencies of cellulose binding domains CBM2 and CBM3 toward cellulose gauze; binding efficiencies of collagen binding domains α1 and α2, chitosan binding domain CBM5, and alginate binding domain VbCBMxx toward respective hydrogel materials.
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]