大肠杆菌DNA复制相关蛋白PriC的初步研究

目的：对大肠杆菌DNA复制相关蛋白PriC进行研究。方法：构建重组表达载体pET21a-PriC及pET21a-PriC1-101,并进一步诱导表达纯化。利用Pulldown手段将pET21a-PriC-notag与pET28at-plus-PriB分别转化到大肠杆菌BL21（DE3）中,诱导表达及共纯化后检测PriC与PriB的相互作用情况。结果：PriC经Ni柱亲和层析纯化获得的蛋白稳定性较差,易沉淀。根据二级结构和蛋白稳定性预测及限制性蛋白酶水解结果,构建了PriC的截短突变蛋白PriC1-101,经Ni亲和层析和superdex75纯化,获得了稳定性较好的蛋白,经Western blot鉴定为目的蛋白。PriC1-101的圆二色谱分析发现其具有较多的α-helix和无规卷曲,这为更好的了解其结构奠定了重要的基础。通过Pulldown手段发现PriC与PriB存在相互作用,并且PriC的稳定性有所提高。结论：PriC蛋白的C-端氨基酸序列是PriC蛋白与PriB蛋白相互作用的主要区域,并对PriC蛋白的稳定性有较大影响。

Preliminary Study of Protein PriC Involved in DNA Replication of Escherichia Coli

Objective：To investigate the PriC,a protein involved in DNA replication of Escherichia coli.Methods：Recombination expression plasmid,pET21a-PriC and pET21a-PriC1-101 were constructed and further induced,expressed and purified.In order to investigate the interaction between PriC and PriB,pET21a-PriC-notag and pET28at-plus-PriB were transformed into Escherichia coli,BL21（DE3） respectively,and induced for expression and purification with the method of Pulldown.Results：The protein of PriC obtained through the purification of Ni chelating sepherose was instable and easily precipitated.According to the secondary structure,protein stability prediction and the results of limited proteolysis experiment, A truncated mutant protein PriC1-101 of PriC was constructed.Proteins with good stability were obtained after purified by Ni chelating sepherose and superdex75,which was identified by Western blot assay as the target protein.PriC1-101 was basically consisted of many α-helix and random coils by circular dichroism detection,which laid an important foundation for a better understanding of the structure.The interaction was detected between PriC and PriB through the Pulldown method and the stability of PriC was improved.Conclusion：C-terminal amino acid sequence of PriC is identified as the main area where PriC interacted with PriB,which has a greater impact on the stability of PriC.