Yang Xiao Ang, Dong Xue Yuan, Li Yan, Wang Yue Dan, Chen Wei Feng
Department of Immunology, School of Basic Medical Science, Peking University Health Science Center, 38 Xue Yuan Road, Beijing 100083, China.
Protein Expr Purif. 2004 Feb;33(2):332-8. doi: 10.1016/j.pep.2003.10.006.
BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy. To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli. The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer. Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97%. The yield of the purified protein was about 78%. The purified recombinant protein was refolded in a simple way. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The recovery rate of refolded protein was 92.1%. The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting. This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods.
BJ-HCC-2是癌胚抗原之一，可能是肿瘤免疫治疗最有前景的靶点。为了研究BJ-HCC-2蛋白在肿瘤细胞中的表达及其引发CTL反应的能力，BJ-HCC-2重组蛋白在大肠杆菌的包涵体中表达。包涵体在碱性缓冲液中用0.3%的N-月桂酰肌氨酸有效溶解。在此变性形式下，携带6x组氨酸标签的BJ-HCC-2蛋白通过Ni-NTA亲和层析一步纯化，纯度超过97%。纯化蛋白的产量约为78%。纯化的重组蛋白以简单的方式复性。通过圆二色性和荧光发射光谱评估，重组蛋白二级和三级结构的恢复验证了其正确复性。复性蛋白的回收率为92.1%。复性后的蛋白通过蛋白质印迹法显示出与BJ-HCC-2蛋白抗体的免疫反应性。这种蛋白质纯化和复性方法易于操作，可能适用于无法通过其他方法纯化的疏水蛋白。
