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

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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)

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