| 杨少媛,朱嘉,吴彦,王晓锐,吴海静,贾宗超,郑积敏.化学通报,2017,80(1):89-93. |
| 大肠杆菌MinC/FtsZ蛋白复合物的纯化和结晶 |
| Purification and Crystallization of MinC/FtsZ Complex in Escherichia coli |
| 投稿时间：2016-09-12 修订日期：2016-10-13 |
| DOI： |
| 中文关键词: 细胞分裂 复合物 结晶 MinC FtsZ |
| 英文关键词:Cell division, Complex, Crystallization, MinC, FtsZ |
| 基金项目:国家自然科学基金(批准号21133003, 21273023)和国家“973”计划项目(批准号2012CB910304)资助。 |
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| 中文摘要: |
| 大肠杆菌在细胞分裂时,FtsZ (Filamentous temperature-sensitive protein Z) 蛋白会在细胞中部潜在位点聚合形成Z环,而MinC蛋白会抑制Z 环形成,从而控制细胞分裂。本研究将FtsZ与MinC目的基因克隆到合适的载体中,并导入到大肠杆菌中进行表达,采用亲和层析和分子筛纯化的方法得到MinC/FtsZ复合物蛋白进行晶体筛选。通过FtsZ、MinC共转化法和FtsZ、MinC分别单独转化、表达纯化后混合两种方法得到FtsZ/MinC蛋白复合物,并分别对其进行晶体筛选。实验结果表明,在适宜的表达条件下,利用分别转化、纯化再混合的方法得到的FtsZ和MinC蛋白复合比例约为1:1；混合时加入GTP和MgCl2可以促进复合物聚集态更单一,通过晶体筛选初步得到形状为针状的FtsZ/MinC复合蛋白晶体,为MinC/FtsZ复合物的结构解析提供实验基础。 |
| 英文摘要: |
| In Escherichia coli, cell division occurs when Z-ring forms at the mid-site of cell through polymerization of FtsZ. The Z-ring formation is inhibited by MinC, resulting in regulation of cell division. Herein, minC and ftsZ were cloned into vectors containing different antibiotics to express recombinant proteins, then MinC and FtsZ were purified by affinity chromatography and gel filtration chromatography. Two different methods, co-transformation, co-expression and co-purification or binding MinC and FtsZ after being expressed and purified respectively, were tried to gain MinC/FtsZ complex for crystal screen. Results showed that with appropriate conditions, through the way of binding MinC and FtsZ after being expressed and purified respectively, MinC/FtsZ complex were in the proportion of 1:1. The addition of GTP and MgCl2helped improve the protein state of MinC/FtsZ complex and needle shape of MinC/FtsZ complex was observed after crystal screen. This study provides experimental basis for structural analysis of MinC/FtsZ complex. |
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