铜离子对绵羊PrP^C重组蛋白结构转化的影响

Effect of copper on the conformational changes of recombinant ovine PrP^C protein

为探明铜离子在朊蛋白构象转化中的作用机制,进一步研究构象病发生机制提供基础数据,利用DNA重组技术,绵羊朊病毒蛋白在大肠杆菌BL21(DE3)中高效表达,获得绵羊PrPC重组蛋白(OvPrPC),与氯化铜在体外结合。对CuCl2处理后的蛋白进行圆二色谱(CD)和蛋白酶K(PK)消化试验进行转化调节后蛋白抗性分析。结果表明,OvPrPC分子量为25172.80u,其二级结构主要由α-螺旋构成,含少量β-折叠。经过CuCl2体外调节后,OvPrPC结构中α-螺旋减少,β-折叠增加。而蛋白酶K消化后,CuCl2作用后的蛋白产生了PK抗性。为进一步的神经细胞毒性试验提供了宝贵的材料,进而为探讨构象病发生机理提供有力的科学依据。

A recombinant ovine prion protein PrP23-256 （OvPrP^C） was expressed in E.coli. and purified using the Ni-NTA agarose. This protein was used for studies of conformational conversion in the presence of copper （Ⅱ） chloride using circular dichroism （CD） spectrum. A structural change characterised by a decreased α -helix and an increased β -sheet was observed in OvPrPc following the CuCl2 treatment. The Copper treatment at physiological condition also made OvPrP23-256 adopt protease-resistant and β-sheet rich conformation. Together, our study demonstrated that Cu^2＋ can dramatically modulate conformational conversion of OvPrP23-256, which possesses protease resistance and β -sheet rich phenotype. These results provide new insight into the pathology ofprion diseases.