Advanced

Expression of recombinant phosphorylated Amelogenin in E.coli using amber codon engineering

Blomgren, Linnea LU (2020) KBKM05 20192
Pure and Applied Biochemistry
Theoretical Chemistry
Abstract
Amelogenin is a multifaced protein, playing an essential role in the formation of enamel where it directs the biomineralisation of hydroxyapatite. The self-assembling properties of amelogenin are highly applicable in the development of novel biomaterials and drug-delivery platforms, and the regenerative properties of the protein are today widely explored in clinical dentistry and wound healing. Using Escherichia coli to express recombinant amelogenin has been a popular choice amongst the amelogenin research community ever since it was first introduced in 1994. One problem with this method is that E. coli lacks the machinery to phosphorylate amelogenin at serine-16; the phosphoserine is believed to be of great mechanistic importance to the... (More)
Amelogenin is a multifaced protein, playing an essential role in the formation of enamel where it directs the biomineralisation of hydroxyapatite. The self-assembling properties of amelogenin are highly applicable in the development of novel biomaterials and drug-delivery platforms, and the regenerative properties of the protein are today widely explored in clinical dentistry and wound healing. Using Escherichia coli to express recombinant amelogenin has been a popular choice amongst the amelogenin research community ever since it was first introduced in 1994. One problem with this method is that E. coli lacks the machinery to phosphorylate amelogenin at serine-16; the phosphoserine is believed to be of great mechanistic importance to the protein. In this study, E. coli expression, in combination with amber codon suppression, was tested as a novel method for production of phosphorylated amelogenin (SepAMG). Four amelogenin isoforms rh174Sep16, rh163Sep16, rh146Sep16 and 104Sep16 were UAG mutated at serine-16, enabling the incorporation of phosphorylated serine via reassignment of the amber stop codon (UAG), when expressed in the presence of an orthogonal translation system (SepOTS). Several expression attempts were made at various induction OD, media composition and SepOTS variants. A selection of samples was concentrated using either ultrafiltration or resuspension of freeze-dried samples, to assess low protein expression levels. No amelogenin expression could be detected when analysed using SDS-PAGE or Western Blot. Expression of the positive control (UAG-mutated green fluorescent protein) was confirmed using fluorimetry. (Less)
Popular Abstract
The enamel covering the teeth crown is the hardest tissue in the body. During tooth development, a protein called amelogenin forms a temporary scaffold which directs and supports the enamel formation. The protein is highly interesting in a scientific point of view. Not only because it holds the key to understanding various forms of tooth deformities, but also because of its self-assembling properties. Amelogenin molecules can easily bind to each other, creating larger protein structures that can be used in the development of novel biomaterials.
Besides extracting amelogenin directly from unerupted animal teeth, a popular alternative used by scientists is to produce the protein in bacteria, usually in Escherichia coli. The problem with... (More)
The enamel covering the teeth crown is the hardest tissue in the body. During tooth development, a protein called amelogenin forms a temporary scaffold which directs and supports the enamel formation. The protein is highly interesting in a scientific point of view. Not only because it holds the key to understanding various forms of tooth deformities, but also because of its self-assembling properties. Amelogenin molecules can easily bind to each other, creating larger protein structures that can be used in the development of novel biomaterials.
Besides extracting amelogenin directly from unerupted animal teeth, a popular alternative used by scientists is to produce the protein in bacteria, usually in Escherichia coli. The problem with that is that bacterial cells and animal cells differ quite a bit in how they process proteins, particularly in how they fold and modify them. In the case of amelogenin, a phosphate group is attached to its amino acid serine-16 when produced in animal cells. However, E. coli cells lack the machinery to perform this type of modification - also called phosphorylation. Although, with the help of a technique called “amber codon suppression”, a synthetic phosphorylation machinery can be implemented into a genetically recoded E. coli strain. This technique was explored in this study as a method to express phosphorylated amelogenin in E. coli.

All organisms on the planet share more or less the same “instruction manual” for how to translate genes into amino acid chains - also known as proteins. Each gene is sectioned into DNA triplet-codons where each codon corresponds to one of the 20 standard amino acids found in nature. Except for three “stop-codons” which mark the end of a gene. In Amber codon suppression, one of these stop-codons, called Amber and coded by UAG, is reassigned to incorporate a non-standard amino acid – in this study a phosphorylated serine. The process of doing so is very complex. It requires both the implementation of an orthogonal translation system and an E. coli strain with all its UAG codons removed from its DNA.

In this study, several experiments were carried out, trying to produce phosphorylated amelogenin using amber codon suppression. Unfortunately, there were no immediate signs of success, which could mean two things. 1. Amelogenin was not expressed at all. 2. Amelogenin was expressed but in amounts below the detection limit of the equipment used in this study. A positive sign, strengthening alternative two is that expression of another protein called GFP, using the same experimental setup, was confirmed by its emission of green fluorescent light. These results encourage further investigation and optimisation of the expression protocol. (Less)
Please use this url to cite or link to this publication:
author
Blomgren, Linnea LU
supervisor
organization
alternative title
Användning av amber codon suppression för produktion av fosforylerat rekombinant amelogenin i E. coli.
course
KBKM05 20192
year
type
H2 - Master's Degree (Two Years)
subject
keywords
amber codon suppression, amelogenin, E. coli, phosphorylation, orthogonal translation system, recombinant protein, applied biochemistry, tillämpad biokemi
language
English
id
9013953
date added to LUP
2020-06-12 13:49:01
date last changed
2020-06-12 13:49:01
@misc{9013953,
  abstract     = {Amelogenin is a multifaced protein, playing an essential role in the formation of enamel where it directs the biomineralisation of hydroxyapatite. The self-assembling properties of amelogenin are highly applicable in the development of novel biomaterials and drug-delivery platforms, and the regenerative properties of the protein are today widely explored in clinical dentistry and wound healing. Using Escherichia coli to express recombinant amelogenin has been a popular choice amongst the amelogenin research community ever since it was first introduced in 1994. One problem with this method is that E. coli lacks the machinery to phosphorylate amelogenin at serine-16; the phosphoserine is believed to be of great mechanistic importance to the protein. In this study, E. coli expression, in combination with amber codon suppression, was tested as a novel method for production of phosphorylated amelogenin (SepAMG). Four amelogenin isoforms rh174Sep16, rh163Sep16, rh146Sep16 and 104Sep16 were UAG mutated at serine-16, enabling the incorporation of phosphorylated serine via reassignment of the amber stop codon (UAG), when expressed in the presence of an orthogonal translation system (SepOTS). Several expression attempts were made at various induction OD, media composition and SepOTS variants. A selection of samples was concentrated using either ultrafiltration or resuspension of freeze-dried samples, to assess low protein expression levels. No amelogenin expression could be detected when analysed using SDS-PAGE or Western Blot. Expression of the positive control (UAG-mutated green fluorescent protein) was confirmed using fluorimetry.},
  author       = {Blomgren, Linnea},
  keyword      = {amber codon suppression,amelogenin,E. coli,phosphorylation,orthogonal translation system,recombinant protein,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Expression of recombinant phosphorylated Amelogenin in E.coli using amber codon engineering},
  year         = {2020},
}