Production and Characterization of Recombinant Amelogenin Phosphorylation Mimics
(2016) KBKM01 20151Pure and Applied Biochemistry
- Abstract
- Amelogenin is an extracellular matrix protein which has an important role to play in enamel formation during tooth development. The need for amelogenin for research purposes in order to be utilized for its many potential applications have resulted in the rise of the production of recombinant amelogenin. So far, the most optimal expression system used for the production of recombinant amelogenin is Escherichia coli. The only difference between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack methionine at the N-terminus and phosphate on Serine-16.The aim of this study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation on amelogenin. A... (More)
- Amelogenin is an extracellular matrix protein which has an important role to play in enamel formation during tooth development. The need for amelogenin for research purposes in order to be utilized for its many potential applications have resulted in the rise of the production of recombinant amelogenin. So far, the most optimal expression system used for the production of recombinant amelogenin is Escherichia coli. The only difference between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack methionine at the N-terminus and phosphate on Serine-16.The aim of this study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation on amelogenin. A total of 16 recombinant amelogenin phosphorylation mimics were successfully constructed by site-directed mutagenesis where serine-16 was mutated to either aspartic acid or glutamic acid. There were difficulties encountered during the purification of the higher positively charged recombinant amelogenin phosphorylation mimics which indicated that factors other than optical density may have a vital effect on their production. No effect of phosphorylation was found on solubility as the phosphorylated amelogenins shared similar solubility profiles to their non-phosphorylated analogues on the pH range of 4 to 8. The solubility of the recombinant amelogenin fusion and phosphorylation mimics were greater than that of the recombinant native amelogenin and its phosphorylation mimic analogue. The analysis of the recombinant amelogenin phosphorylation mimics by dynamic light scattering showed that they too can form nanospheres. There were minor variations in hydrodynamic radii and polydispersity indices of the recombinant phosphorylated and non-phosphorylated amelogenins at both 20oC and 37oC which indicated that phosphorylation had not affected amelogenin self-assembly. It was found that the recombinant amelogenin phosphorylation mimics as well as their non-phosphorylated analogues had similar strong apatite-binding affinity which also showed no effect of phosphorylation on the binding affinity of amelogenin. (Less)
- Popular Abstract
- We use our teeth daily to eat all sorts of food with varying degrees of texture and hardness. The white part of our teeth called the enamel is the hardest tissue of the human body. Have you ever wondered what makes our teeth so hard and strong that we can chew on a chicken bone? Well, the answer is amelogenin which is the main subject of this study. Amelogenin is one of the many proteins that help build the enamel. However, amelogenin is of greatest importance to enamel formation because in its absence scientists have found that it leads to teeth defects.
Scientists have also discovered that amelogenin can be utilized in many applications such as teeth defects, wound healing, bone formation and regeneration and many others. So far, there... (More) - We use our teeth daily to eat all sorts of food with varying degrees of texture and hardness. The white part of our teeth called the enamel is the hardest tissue of the human body. Have you ever wondered what makes our teeth so hard and strong that we can chew on a chicken bone? Well, the answer is amelogenin which is the main subject of this study. Amelogenin is one of the many proteins that help build the enamel. However, amelogenin is of greatest importance to enamel formation because in its absence scientists have found that it leads to teeth defects.
Scientists have also discovered that amelogenin can be utilized in many applications such as teeth defects, wound healing, bone formation and regeneration and many others. So far, there are two amelogenin-based products on the market. These products include (i) Emdogain® for the treatment of periodontitis, an enamel defect due to lack of proper amelogenin and (ii) Xelma® used to treat leg ulcers. As a result of the numerous potential applications of amelogenin, thus, there is increasingly more research carried out on amelogenins.
So far, the most optimal production system used for the production of recombinant amelogenin is the bacteria called Escherichia coli. The only difference that existed between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack the amino acid methionine at the N-terminus and phosphate group on Serine-16.The aim of this present study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation of amelogenin on its properties. The properties of the novel phosphorylated amelogenin mimics with that of non-phosphorylated amelogenins were compared in order to examine the effects of the phosphorylation. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8872145
- author
- Jean Baptiste, Krishma Eloise LU
- supervisor
- organization
- course
- KBKM01 20151
- year
- 2016
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- tillämpad biokemi, applied biochemistry
- language
- English
- id
- 8872145
- date added to LUP
- 2016-05-12 13:36:59
- date last changed
- 2016-05-12 13:36:59
@misc{8872145,
abstract = {{Amelogenin is an extracellular matrix protein which has an important role to play in enamel formation during tooth development. The need for amelogenin for research purposes in order to be utilized for its many potential applications have resulted in the rise of the production of recombinant amelogenin. So far, the most optimal expression system used for the production of recombinant amelogenin is Escherichia coli. The only difference between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack methionine at the N-terminus and phosphate on Serine-16.The aim of this study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation on amelogenin. A total of 16 recombinant amelogenin phosphorylation mimics were successfully constructed by site-directed mutagenesis where serine-16 was mutated to either aspartic acid or glutamic acid. There were difficulties encountered during the purification of the higher positively charged recombinant amelogenin phosphorylation mimics which indicated that factors other than optical density may have a vital effect on their production. No effect of phosphorylation was found on solubility as the phosphorylated amelogenins shared similar solubility profiles to their non-phosphorylated analogues on the pH range of 4 to 8. The solubility of the recombinant amelogenin fusion and phosphorylation mimics were greater than that of the recombinant native amelogenin and its phosphorylation mimic analogue. The analysis of the recombinant amelogenin phosphorylation mimics by dynamic light scattering showed that they too can form nanospheres. There were minor variations in hydrodynamic radii and polydispersity indices of the recombinant phosphorylated and non-phosphorylated amelogenins at both 20oC and 37oC which indicated that phosphorylation had not affected amelogenin self-assembly. It was found that the recombinant amelogenin phosphorylation mimics as well as their non-phosphorylated analogues had similar strong apatite-binding affinity which also showed no effect of phosphorylation on the binding affinity of amelogenin.}},
author = {{Jean Baptiste, Krishma Eloise}},
language = {{eng}},
note = {{Student Paper}},
title = {{Production and Characterization of Recombinant Amelogenin Phosphorylation Mimics}},
year = {{2016}},
}