Expanded Codon Technology for Producing Phosphorylated Enamel Matrix Protein

Abstract

Amelogenin, a critical protein in enamel formation, self-assembles into supramolecular structure that guide hydroxyapatite (HAP) crystal growth that is essential for the development of strong and healthy enamel. Phosphorylation of amelogenin at Ser-16 plays a pivotal role in modulating its self-assembly and functional properties, impacting the overall enamel biomineralization process. This post-translational modification (PTM) enhances the ability of the protein to interact with calcium ions, which are crucial for HAP nucleation and growth. Production of recombinant amelogenin using prokaryotic system results in a protein that does not undergo PTM including phosphorylation. Recent advancements in genetic code expansion (GCE), particularly the third generation (pSer-3G), have enabled the incorporation of non-canonical amino acids (ncAAs) at specific site. These advancements have made it possible to produce phosphorylated recombinant amelogenin with high percentage of phosphoserine incorporation. In the previous study we have successfully constructed recombinant plasmids that can express phosphorylated human amelogenin in prokaryotic system using this third-generation genetic code expansion.
This study aimed to optimize the production of several phosphorylated recombinant human amelogenin constructs and investigate the effects of amino acid mutations on their self-assembly properties. The constructs included full-length amelogenin (rH174-sep16) and its cleavage product (rH146-sep16), as well as cleavage products with mutations (rH146-sep16-T37I and rH146-sep16-P56T) related to the genetic disease Amelogenesis Imperfecta (AI). Optimization was focused on cultivation media, inducer concentration, point of induction, duration of induction, and temperature. The results indicated that Terrific Broth (TB) media with 0.25 mM IPTG induction at an optical density (OD) of 4.5 provided the highest protein yield. However, protein phosphorylation levels were higher when the induction was conducted at OD600 1.5, as confirmed by Phos-tag SDS-PAGE. Mass spectrometry confirmed the presence of phosphorylated amelogenin; however, the signal cannot be used to quantify the phosphorylation level accurately due to the fact that phosphorylated proteins are tend to be more difficult to ionize.
Self-assembly studies showed that rH146-sep16 could form nanoribbon structures in the presence of calcium and phosphate ions. The P56T mutation allowed for the formation of these structures, though in reduced quantities, while the T37I mutation inhibited nanoribbon formation. These findings highlight the different impacts of specific mutations on the ability of amelogenin to self-assemble.

Popular Abstract

How Scientists Are Cracking the Code to Stronger Teeth
Did you know that the secret to strong, healthy teeth lies in a special protein called amelogenin? This protein is like a tiny architect, creating the perfect conditions for enamel, the hard outer layer of our teeth, to grow. But here's the twist: amelogenin needs a little boost in the form of a phosphate molecule at a specific spot (Ser-16) to do its job really well. This boost helps it grab onto calcium, which is crucial for building strong enamel.
Normally, when scientists make amelogenin in the lab using bacteria, it misses out on this crucial phosphate. But thanks to some cutting-edge genetic engineering tricks, researchers can now produce a version of amelogenin with this phosphate in just the right place. This study was all about fine-tuning the production of this supercharged amelogenin and figuring out how small changes in its structure affect its ability to build enamel.
To get the best results, scientists experimented with different methods, tweaking everything from the growth media to the timing and amount of a chemical that kicks off protein production. They discovered that using a special growth broth and adding the chemical inducer at the perfect moment resulted in the highest yield of this important protein. They confirmed it was phosphorylated, though measuring the exact amount was a bit tricky.
In their experiments, they found that the phosphorylated part of amelogenin (rH146-sep16) could form tiny, ribbon-like structures when mixed with calcium and phosphate – exactly what’s needed for enamel to grow. Interestingly, a mutation (P56T) slightly reduced these structures, while another mutation (T37I) stopped them completely. This gives us valuable insights into how changes in amelogenin can affect enamel formation and help us understand conditions like Amelogenesis Imperfecta, a genetic disorder that affects tooth enamel.
In short, by cracking the code of how amelogenin works and how to produce it correctly in the lab, scientists are paving the way for better treatments for tooth enamel disorders and possibly even stronger, healthier teeth for everyone!

• author: Aji, Nurul Apsari
• supervisor: Johan Svensson Bonde
• organization: Pure and Applied Biochemistry
• course: KBKM01 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Amelogenin, Phosphorylation, genetic code expansion, applied biochemistry
• language: English
• id: 9163349
• date added to LUP: 2024-06-14 14:02:38
• date last changed: 2024-06-14 14:02:38