RESULTS: In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml.
CONCLUSIONS: These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.
结果:在研究中,人釉质的32kDa片段在大肠杆菌中成功表达,可以使用固定化金属离子亲和层析纯化(IMAC)获得,透析,和冻干。该工作流程导致每升培养物大约10mg的enamelin的产量。使用Ni2作为金属离子获得了IMAC纯化的最佳条件,并且当在结合和洗涤步骤期间包括30mM咪唑时。此外,体外矿化实验表明,重组烯酰胺在0.5mg/ml的浓度下可以促进磷酸钙矿化。
结论:这些发现通过促进釉质形成机制的进一步研究来解决釉质的稀缺性,并为开发釉质启发的矿化生物材料开辟了新的途径。