Cytoplasmic expression, antibody production, and characterization of the novel zinc finger protein 637
Cytoplasmic expression, antibody production, and characterization of the novel zinc finger protein 637
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第637章 新型锌指蛋白的细胞质表达、抗体产生和表征
DOI：
10.1007/s00253-012-4235-5
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发表时间：
2012-06
影响因子：
5
通讯作者：
Wei, Yuquan
中科院分区：
文献类型：
--
作者：
Li, Kai;Wei, Yuyan;Zhang, Jie;Li, Jun;Gao, Bo;Huang, Lugang;Lin, Ping;Wei, Yuquan
锌指蛋白637(Zfp637)属于Kruppel样蛋白家族，由一个非典型C2H2和六个连续的典型锌指基序组成。根据zfp637的结构特征及其在细胞核中的定位，我们推测zfp637可能作为一种DNA结合蛋白来调节基因转录。然而，到目前为止，由于缺乏纯化的zfp637蛋白和任何商业抗体来检测细胞和组织中的zfp637蛋白，限制了zfp637的功能研究。在此，我们建立并优化了一种融合基因zfp637和谷胱甘肽转移酶(GST)的表达系统，以获得最高产量的可溶性GST-zfp637融合蛋白。产量约为原细菌培养物的10 mg/L。纯化重组GST-zfp637融合蛋白，用于兔多克隆抗体的制备。此外，我们开发了一种去除抗GST抗体成分的方法，并获得了高纯度的抗zfp637抗体，通过酶联免疫吸附试验证明。Western blotting结果表明，抗zfp637抗体不仅能识别重组的zfp637蛋白，还能识别多种细胞株的内源性zfp637蛋白。免疫荧光和免疫组织化学结果显示该蛋白主要定位于细胞核。200μM的H_2O_2处理NIH_3T_3细胞后，Zfp637m RNA和蛋白的表达水平显著升高，并呈时间依赖性。重组GST-zfp637融合蛋白和我们纯化的抗zfp637抗体将有助于阐明zfp637的功能。
Zinc finger protein 637 (zfp637), belonging to the Kruppel-like protein family, comprises one atypical C2H2and six consecutive typical zinc finger motifs. Based on the structural characterization of zfp637 and its location in the cell nucleus, we predict that zfp637 might function as a DNA-binding protein to regulate gene transcription. However, the absence of both a purified zfp637 protein and any commercial antibody for detecting it in cells and tissues has limited functional studies of zfp637 to date. Here, we developed and optimized an expression system by fusing zfp637 with glutathioneS-transferase (GST) to achieve a maximal yield of soluble GST-zfp637 fusion protein inEscherichia coliBL21(DE3) cells. The yield was about 10 mg/l of the original bacterial culture. The recombinant GST-zfp637 fusion protein was purified and used for polyclonal antibody production in rabbits. In addition, we developed a method to remove the anti-GST antibody component and obtained a highly purified anti-zfp637 antibody, as demonstrated by an enzyme-linked immunosorbent assay. Western blotting showed that the anti-zfp637 antibody recognized not only the recombinant zfp637 protein but also endogenous zfp637 in several cell lines. The protein was localized mainly in the cell nucleus by immunofluorescence and immunohistochemistry. The expression levels of zfp637 mRNA and protein were significantly increased in NIH3T3 cells treated with 200 μM of H2O2in a time-dependent manner. The recombinant GST-zfp637 fusion protein and our purified anti-zfp637 antibody will help in elucidating the function of zfp637.
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DOI：
10.1016/j.bbrc.2009.07.079
发表时间：
2009-09
影响因子：
3.1
作者：
Hye-Min Park;Goo-Young Kim;Min-Kyung Nam;Geun-Hye Seong;Chul Han;K. Chung;Seongman Kang;H. Rhim
通讯作者：
Hye-Min Park;Goo-Young Kim;Min-Kyung Nam;Geun-Hye Seong;Chul Han;K. Chung;Seongman Kang;H. Rhim
影响因子：
6.4
作者：
Schultz, Tina;Martinez, Lucia;de Marco, Ario
通讯作者：
de Marco, Ario
DOI：
10.1523/jneurosci.23-09-03597.2003
发表时间：
2003-05
期刊：
The Journal of Neuroscience
影响因子：
--
作者：
H. Ryu;Junghee Lee;K. Zaman;James Kubilis;R. Ferrante;B. Ross;R. Neve;R. Ratan
通讯作者：
H. Ryu;Junghee Lee;K. Zaman;James Kubilis;R. Ferrante;B. Ross;R. Neve;R. Ratan
影响因子：
3.5
作者：
Deschênes, F;Massip, L;Lebel, M
通讯作者：
Lebel, M
DOI：
--
发表时间：
2005
期刊：
--
影响因子：
--
作者：
通讯作者：
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