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Creating protein aggregates with the ability to form thiol-specific covalent bonds through genetic modification of amelogenin

Persson, Fredrik LU (2017) KBK820 20171
Pure and Applied Biochemistry
Abstract
Amelogenin is an enamel matrix protein which participates in the formation of dental enamel, the hardest tissue in the human body. The protein participates in this process by orienting the mineral crystals which make up the enamel and several proteolytic products from amelogenin also affect the dental enamel during its maturation. The protein is mostly hydrophobic with a hydrophilic region at the C-terminal and is regarded as an intrinsically disordered protein (IDP) due to its lack of secondary and tertiary structure. In addition to being an IDP the protein is capable of self-aggregating in order to create larger structures. One of these structures is the nanosphere. The formation of these spheres is highly pH-dependant and affects the... (More)
Amelogenin is an enamel matrix protein which participates in the formation of dental enamel, the hardest tissue in the human body. The protein participates in this process by orienting the mineral crystals which make up the enamel and several proteolytic products from amelogenin also affect the dental enamel during its maturation. The protein is mostly hydrophobic with a hydrophilic region at the C-terminal and is regarded as an intrinsically disordered protein (IDP) due to its lack of secondary and tertiary structure. In addition to being an IDP the protein is capable of self-aggregating in order to create larger structures. One of these structures is the nanosphere. The formation of these spheres is highly pH-dependant and affects the solubility of the protein with the protein being in its monomer form under acidic conditions and in the aggregate form under alkaline conditions. These nanospheres are of great interest to the scientific community since it has been shown that by producing fusion proteins containing amelogenin it is possible to create nanospheres with a wide range of abilities. The purpose of this project is to synthesize fusion proteins with amelogenin and a cysteine residue bound together by a linker in order to determine if it is possible to create fusion proteins with similar properties as the wild type protein while also being capable of specifically binding molecules to the cysteine residue. Furthermore the feasibility of a model where maleimide derivatives are used as a way to bind molecules with different properties to the nanosphere will be investigated. This project has shown that it is possible to create amelogenin+ cysteine fusion proteins with similar properties as the wild type protein if a reducing agent is present. The need for a reducing agent is due to formation of disulphide bridges between cysteines on different nanospheres which causes excessive aggregation. In addition to the properties of the wild type amelogenin the availability of the cysteine residues for reactions has been shown. The feasibility of the maleimide derivative model is still unknown due to the unsuitable derivative used in this study. Further research is needed for the characterization of the proteins and finding a suitable molecule to test the maleimide derivative model. (Less)
Popular Abstract
Amelogenin is a protein that can be produced by several species, including humans and is capable of gathering together in water to form microscopic aggregates called nanospheres. In this master thesis the DNA sequence of amelogenin was altered so that different fusion proteins were created. These fusion proteins differ from normal amelogenin in that the fusion protein aggregates have thiols, chemical molecules consisting of sulphur and hydrogen on the aggregate surface. The thiols is a result of the fusion proteins containing a specific amino acid called cysteine. Amino acids are molecules that cells use to produce proteins and cysteine is often used by cells to give proteins stability. This stability is a result of the thiols on the... (More)
Amelogenin is a protein that can be produced by several species, including humans and is capable of gathering together in water to form microscopic aggregates called nanospheres. In this master thesis the DNA sequence of amelogenin was altered so that different fusion proteins were created. These fusion proteins differ from normal amelogenin in that the fusion protein aggregates have thiols, chemical molecules consisting of sulphur and hydrogen on the aggregate surface. The thiols is a result of the fusion proteins containing a specific amino acid called cysteine. Amino acids are molecules that cells use to produce proteins and cysteine is often used by cells to give proteins stability. This stability is a result of the thiols on the cysteines being able to react with one another if they are close enough and create a strong bond called a disulphide bridge.
The purpose of this study was to produce and purify different fusion proteins consisting of amelogenin and cysteines and then compare some of the properties of the fusion proteins with ordinary amelogenin. After this a model was tested where a group of molecules called maleimide derivatives can be used to alter the properties of the fusion protein nanospheres. Maleimide derivatives were used in this model since they react specifically with thiols under the right conditions.
When the proteins had been produced and purified five experiments were performed. First an SDS-PAGE was performed with both non-purified and pure protein samples where the samples were injected into a gel and analysed in order to see if the proteins had been produced and if they were pure. After that the solubility and aggregation of the fusion proteins were compared with amelogenin. The solubility was tested with solubility profiles where protein samples in water at different pH were prepared and analysed with the same type of gel as in SDS-PAGE. The aggregation was tried with DLS which is an experiment where a sample is analysed with a laser and depending on how the sample affects the scattering of the light from the laser the size of the aggregates in the sample can be determined. Thereafter the reactivity of the thiols on the nanospheres were tested by measuring how much light each protein absorbs in a specific reaction. Finally the possibility of using maleimide derivatives to alter the properties of fusion protein nanospheres was tested by using a derivative that fluoresces when it binds to thiols.

The experiments showed that the fusion proteins could be produced and purified. Regarding the properties of the new proteins the solubility and aggregation experiments indicated that the nanospheres formed unwanted disulphide bridges between each other resulting in larger aggregates. These disulphide bridges could be broken with a reducing agent resulting in the fusion proteins having similar solubility and aggregation as amelogenin. The third property that was tested was thiol reactivity and the absorbance measurements showed that almost all fusion proteins had reactive cysteines. The exception was one protein that was likely less water soluble than the others which affected the measurement. In the end the maleimide derivative model was tested with the fluorescent molecule but unfortunately the derivative was unspecific and likely reacted with the nanospheres in unwanted ways since the molecule reacted with nanospheres made up of normal amelogenin which does not contain thiols.

Even though this study has answered many questions about the fusion proteins made in this study there is a need for more research regarding the properties of the proteins. For example there is a need to find more suitable maleimide derivatives in order to test if they can be used to alter the properties of nanospheres
To conclude it can be said that the project has been successful in producing and purifying the desired proteins. Additionally the performed experiments showed that the fusion proteins had similar properties as common amelogenin if a reducing agent is present and that the thiols on the aggregate surface are reactive. Unfortunately it is still unclear whether maleimide derivatives can be used to modify nanospheres since the molecule chosen for this study was unspecific in its binding. (Less)
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author
Persson, Fredrik LU
supervisor
organization
course
KBK820 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
fusion protein, cysteine, nanosphere, amelogenin, self-aggregation, maleimide derivatives, applied biochemistry, tillämpad biokemi
language
English
id
8908901
date added to LUP
2017-09-18 14:14:23
date last changed
2017-09-18 14:14:23
@misc{8908901,
  abstract     = {Amelogenin is an enamel matrix protein which participates in the formation of dental enamel, the hardest tissue in the human body. The protein participates in this process by orienting the mineral crystals which make up the enamel and several proteolytic products from amelogenin also affect the dental enamel during its maturation. The protein is mostly hydrophobic with a hydrophilic region at the C-terminal and is regarded as an intrinsically disordered protein (IDP) due to its lack of secondary and tertiary structure. In addition to being an IDP the protein is capable of self-aggregating in order to create larger structures. One of these structures is the nanosphere. The formation of these spheres is highly pH-dependant and affects the solubility of the protein with the protein being in its monomer form under acidic conditions and in the aggregate form under alkaline conditions. These nanospheres are of great interest to the scientific community since it has been shown that by producing fusion proteins containing amelogenin it is possible to create nanospheres with a wide range of abilities. The purpose of this project is to synthesize fusion proteins with amelogenin and a cysteine residue bound together by a linker in order to determine if it is possible to create fusion proteins with similar properties as the wild type protein while also being capable of specifically binding molecules to the cysteine residue. Furthermore the feasibility of a model where maleimide derivatives are used as a way to bind molecules with different properties to the nanosphere will be investigated. This project has shown that it is possible to create amelogenin+ cysteine fusion proteins with similar properties as the wild type protein if a reducing agent is present. The need for a reducing agent is due to formation of disulphide bridges between cysteines on different nanospheres which causes excessive aggregation. In addition to the properties of the wild type amelogenin the availability of the cysteine residues for reactions has been shown. The feasibility of the maleimide derivative model is still unknown due to the unsuitable derivative used in this study. Further research is needed for the characterization of the proteins and finding a suitable molecule to test the maleimide derivative model.},
  author       = {Persson, Fredrik},
  keyword      = {fusion protein,cysteine,nanosphere,amelogenin,self-aggregation,maleimide derivatives,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Creating protein aggregates with the ability to form thiol-specific covalent bonds through genetic modification of amelogenin},
  year         = {2017},
}