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目的筛选一个新的锌指蛋白Mip1的DNA结合位点。方法将Mip1cDNA全长克隆入原核表达质粒pGEX-4T-1构建了Mip1的原核表达质粒pGEX-Mip1。用谷胱甘肽亲和层析纯化GST-Mip1融合蛋白。通过固定化的GST-Mip1,从寡核苷酸文库中俘获Mip1的结合的寡核苷酸片段;将所得寡核苷酸片段插入T-载体,通过测序、序列比对得到DNA结合位点。结果锌指蛋白Mip1的融合蛋白能在大肠杆菌中高效表达,能与固定化还原性谷胱甘肽很好地亲和而得到纯化;用固定化的Mip1融合蛋白俘获到了其结合的寡核苷酸序列,通过测序、序列比对,得到了Mip1的DNA结合位点。结论Mip1的DNA结合位点的核心序列为G/TCCTA,Mip1的DNA结合位点的成功确定为Mip1功能的深入研究打下了基础。
Objective To screen the DNA binding site of Mip1, a new zinc finger protein. Methods The full length Mip1 cDNA was cloned into the prokaryotic expression vector pGEX-4T-1 to construct the prokaryotic expression vector pGEX-Mip1 of Mip1. GST-Mip1 fusion protein was purified by glutathione affinity chromatography. Mip1-bound oligonucleotide fragments were captured from the oligonucleotide library by immobilized GST-Mip1; the resulting oligonucleotide fragments were inserted into the T-vector and DNA binding sites were obtained by sequencing and sequence alignment. Results The fusion protein of zinc finger protein Mip1 was highly expressed in E.coli and purified well with immobilized reduced glutathione. The immobilized Mip1 fusion protein captured its bound oligonucleotide The sequence of DNA binding site of Mip1 was obtained by sequencing and sequence alignment. Conclusion The core sequence of DNA binding site of Mip1 is G / TCCTA. The successful identification of the Mip1 DNA binding site lays a foundation for the further study of Mip1 function.