Ferritin is a ubiquitous iron storage protein which plays key role in regulating iron homeostasis and metabolism. In this paper, the ferritin heavy chain homologs (HCH) and light chain homologs (LCH) from Bombyx mori (BmFerHCH and BmFerLCH) were amplified through PCR and cloned into the expression vector pET-30a(+). The recombinant BmFerHCH and BmFerLCH expressed in Escherichia coli were in the form of insoluble inclusion bodies, indicating that the two proteins were not in their natural structural conformation. In order to obtain refolded ferritin in vitro, the inclusion bodies (BmFerHCH and/or BmFerLCH) were dissolved in denaturing buffer (100 mM Tris, 50 mM Glycine, 8 M urea, 5 mM DTT, pH 8.0) and then refolded in refolding buffer (100 mM Tris, 400 mM l-arginine, 0.2 mM PMSF, 0.5 mM DTT). The result showed that it was only when both BmFerHCH and BmFerLCH were present together in the denaturing buffer that refolding was successful and resulted in the formation of heteropolymers (H–L chain dimers) over homopolymers (H–H chain or L–L chain dimers). Moreover, the molecules (NaCl, Triton and glycerol) were found to enhance protein refolding. The optimum temperature, pH and ratios of BmFerHCH/BmFerLCH required for refolding were found to be 10 °C, pH 7, 1:1 or 1:2, respectively. Finally, the refolded ferritin had the ability to store iron, exhibited ferroxidase activity, and could withstand high temperatures and pH treatment, which is consistent with ferritin in other species.

铁蛋白是一种普遍存在的铁存储蛋白，在调节铁稳态和新陈代谢中起关键作用。在本文中，由铁蛋白重链的同系物（HCH）和轻链同系物（LCH）家蚕（BmFerHCH和BmFerLCH）通过PCR扩增并克隆入表达载体pET-30a中（+）。在大肠杆菌中表达的重组BmFerHCH和BmFerLCH它们是不溶性包涵体的形式，表明这两种蛋白不在其天然结构构象中。为了在体外获得重折叠的铁蛋白，将包涵体（BmFerHCH和/或BmFerLCH）溶解在变性缓冲液（100 mM Tris，50 mM甘氨酸，8 M尿素，5 mM DTT，pH 8.0）中，然后在重折叠缓冲液中重折叠（100 mM Tris，400 mM  l-精氨酸，0.2 mM PMSF，0.5 mM DTT）。结果表明，只有在变性缓冲液中同时存在BmFerHCH和BmFerLCH时，重折叠才能成功，并导致均聚物（H-H链或L-L链二聚体）上形成杂聚物（H-L链二聚体）。 ）。此外，发现分子（NaCl，Triton和甘油）可增强蛋白质复性。重新折叠所需的最佳温度，pH和BmFerHCH / BmFerLCH的最佳比例分别为10°C，pH 7、1：1或1：2。最后，重新折叠的铁蛋白具有储存铁的能力，表现出铁氧化酶活性，并且可以经受高温和pH处理，这与其他物种中的铁蛋白一致。