Exploring the Potential of Amelogenin-Silk Fusion Proteins as Novel Biomaterials
(2024) KBKM01 20241Pure and Applied Biochemistry
- Abstract
- The advancing field of biomaterials seeks to improve the quality of life by restoring lost biological functions. While the use of naturally or synthetically originating biomaterials poses scalability and toxicity issues, proteins are an interesting alternative characterized by biocompatibility and tunability. Amelogenin and dragline silks are two well-studied proteins of interest due to their mechanical properties and distinct nature of self-assembly, making them unique candidates for biotechnological and medical applications. In this study, the rH174 and rH146 amelogenin variants were fused with major ampullate spidroin 2 (MaSp2) silk with an aim to investigate the formation and stability of the fusion protein assembly and to assess the... (More)
- The advancing field of biomaterials seeks to improve the quality of life by restoring lost biological functions. While the use of naturally or synthetically originating biomaterials poses scalability and toxicity issues, proteins are an interesting alternative characterized by biocompatibility and tunability. Amelogenin and dragline silks are two well-studied proteins of interest due to their mechanical properties and distinct nature of self-assembly, making them unique candidates for biotechnological and medical applications. In this study, the rH174 and rH146 amelogenin variants were fused with major ampullate spidroin 2 (MaSp2) silk with an aim to investigate the formation and stability of the fusion protein assembly and to assess the potential of amelogenin as an enzymatically cleavable tag for purification. Proteolytic digestion with trypsin led to the separation of the MaSp2 domain attached to the C-terminus of the rH174 protein, resulting in the putative identification of protein fragments and failure to retrieve silk from the soluble fraction upon amelogenin precipitation. DLS measurements revealed inconclusive results for proteins incubated in different concentrations of potassium phosphate at acidic pH. Incubation of the samples in methanol at alkaline conditions demonstrated that the 174_2 protein was more likely to form larger, non-precipitating aggregates than the rH146-containing constructs. Protein solubilization upon exposure to potassium phosphate solution at alkaline pH demonstrated the increased stability of 174_2 and 146_2 protein aggregates. The film disintegration assay proved the irreversible nature of silk assembly in the 174_2 construct. The presence of intermolecular β-sheets in silk-containing proteins was initially determined by preliminary studies using the thioflavin T fluorescence assay. To entirely unveil the potential of amelogenin-silk fusion proteins, further investigation is required. (Less)
- Popular Abstract
- Degenerative diseases have a negative impact on human life, impairing different parts of the body. Nowadays, modern medicine seeks for efficient and novel solutions to prevent the occurrence of such disorders. Biomaterials are an interesting alternative to this matter, which consist of substances with properties capable of restoring the function of the damaged area in the human body. One of the sources to create biomaterials is proteins, which can be designed to meet the requirements of specific biomedical applications. This is possible due to the fascinating field of bioengineering, which provides numerous tools for modifying and combining various properties of proteins. Among them, amelogenin is an interesting protein candidate as a... (More)
- Degenerative diseases have a negative impact on human life, impairing different parts of the body. Nowadays, modern medicine seeks for efficient and novel solutions to prevent the occurrence of such disorders. Biomaterials are an interesting alternative to this matter, which consist of substances with properties capable of restoring the function of the damaged area in the human body. One of the sources to create biomaterials is proteins, which can be designed to meet the requirements of specific biomedical applications. This is possible due to the fascinating field of bioengineering, which provides numerous tools for modifying and combining various properties of proteins. Among them, amelogenin is an interesting protein candidate as a biomaterial. It is found in all mammals, including humans, and it participates in the creation of tooth enamel minerals. The main feature of amelogenin is its self-assembly, which leads to the creation of precisely organized structures. In addition, spider silk also has extraordinary properties, characterized by elasticity and mechanical strength, which are desirable from a biomedical point of view.
In this project, amelogenin and silk proteins were combined together to investigate their behavior under different experimental conditions and evaluate their potential as biomaterials for medical purposes. Using various methods offered by molecular biology, four distinct amelogenin-silk proteins were designed and produced in a bacterial expression system using Escherichia coli as a host. The proteins have been characterized using biophysical techniques, mainly aimed at determining their propensity to form aggregates upon exposure to agents that cause the protein assembly, and the tendency of protein films to dissolve. From all the proteins tested, the 174_2 construct proved to be the most promising biomaterial candidate. It was able to form stable aggregates, preventing the complete dissolution of the protein film in a physiological solution. This finding was especially exciting when thinking about the stability of the implanted protein biomaterial to replace the lost biological functionality. On the other hand, constructs containing the rH146 protein tended to disintegrate, making them an unattractive candidate for biomaterials.
Other experiments did not go as planned, leading to inconclusive results. These included the separation of silk protein from amelogenin and the formation of higher-level structures by silk. Unfortunately, they could not be repeated or improved because of time limits. However, this provides an opportunity to continue research on amelogenin-silk proteins to fully discover their potential. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9158793
- author
- Jamróg, Grzegorz LU
- supervisor
- organization
- course
- KBKM01 20241
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Biomaterial, Protein, Amelogenin, Silk, Applied Biochemistry
- language
- English
- id
- 9158793
- date added to LUP
- 2024-06-13 09:50:42
- date last changed
- 2024-06-13 09:50:42
@misc{9158793,
abstract = {{The advancing field of biomaterials seeks to improve the quality of life by restoring lost biological functions. While the use of naturally or synthetically originating biomaterials poses scalability and toxicity issues, proteins are an interesting alternative characterized by biocompatibility and tunability. Amelogenin and dragline silks are two well-studied proteins of interest due to their mechanical properties and distinct nature of self-assembly, making them unique candidates for biotechnological and medical applications. In this study, the rH174 and rH146 amelogenin variants were fused with major ampullate spidroin 2 (MaSp2) silk with an aim to investigate the formation and stability of the fusion protein assembly and to assess the potential of amelogenin as an enzymatically cleavable tag for purification. Proteolytic digestion with trypsin led to the separation of the MaSp2 domain attached to the C-terminus of the rH174 protein, resulting in the putative identification of protein fragments and failure to retrieve silk from the soluble fraction upon amelogenin precipitation. DLS measurements revealed inconclusive results for proteins incubated in different concentrations of potassium phosphate at acidic pH. Incubation of the samples in methanol at alkaline conditions demonstrated that the 174_2 protein was more likely to form larger, non-precipitating aggregates than the rH146-containing constructs. Protein solubilization upon exposure to potassium phosphate solution at alkaline pH demonstrated the increased stability of 174_2 and 146_2 protein aggregates. The film disintegration assay proved the irreversible nature of silk assembly in the 174_2 construct. The presence of intermolecular β-sheets in silk-containing proteins was initially determined by preliminary studies using the thioflavin T fluorescence assay. To entirely unveil the potential of amelogenin-silk fusion proteins, further investigation is required.}},
author = {{Jamróg, Grzegorz}},
language = {{eng}},
note = {{Student Paper}},
title = {{Exploring the Potential of Amelogenin-Silk Fusion Proteins as Novel Biomaterials}},
year = {{2024}},
}