This is achieved by utilizing elements of the unfolded protein response (UPR) ( Tsuchiya et al., 2008) to maintain ameloblast ER proteostasis as commonly seen in other secretory cells ( Moore and Hollien, 2012). Ameloblasts are secretory cells that are adapted to handle the large amount of amelogenin transiting through the rough endoplasmic reticulum (ER) and Golgi apparatus ( Warshawsky, 1968). On secretion, it forms “nanospheres” of ~25–50 nm in diameter in the developing enamel ( Fincham et al., 1995). Wild-type (WT) amelogenin begins to self-assemble during transit through the ameloblast secretory pathway ( Brookes et al., 2006). Our own interest is with the effect of a mouse amelogenin mutation (p.Y64H) on amelogenin aggregation/assembly and ameloblast secretory trafficking. However, the exact function of amelogenin remains unclear and studies are ongoing to elucidate amelogenin function and the etiologies that underpin AI when the amelogenin gene is mutated. Amelogenin mutations can lead to amelogenesis imperfecta (AI) in humans ( Lagerstrom et al., 1991 Aldred et al., 1992 Lench and Winter, 1995 Collier et al., 1997 Kindelan et al., 2000 Hart et al., 2002 Kim et al., 2004 Wright et al., 2009) and mouse models ( Barron et al., 2010) and amelogenin null mice fail to produce enamel ( Gibson et al., 2001 Wright et al., 2009 Smith et al., 2016). Amelogenin, present as a range of related molecules generated through extracellular proteolysis of several alternatively spliced gene products, comprises ~90% of the total extracellular protein matrix and is essential for normal enamel biomineralisation. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.Īmelogenesis involves the incremental secretion of an extracellular protein matrix by ameloblasts. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. ![]() Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Division of Oral Biology, School of Dentistry, University of Leeds, Leeds, United Kingdom.
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