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Abstract：
BACKGROUND: Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent.
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.
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.
摘要：
背景：Enamelin是一种釉质基质蛋白，在釉质的形成中起着至关重要的作用，人体中最矿化的组织。先前使用动物模型和来自天然来源的蛋白质的研究指出，在釉质形成过程中，釉质素在促进矿化事件中的关键作用。然而,天然来源的搪瓷素是稀缺的，因此，通过目前的研究，我们的目的是建立一个简单的微生物生产方法的重组人搪瓷素，以支持其作为矿化剂的使用。
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。
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。
