| 任丽丽,吴伟,施怡,岳凌,杨占山.耐辐射奇球菌PprI蛋白质在毕赤酵母菌中的高效表达及纯化[J].中华放射医学与防护杂志,2016,36(6):406-411 |
| 耐辐射奇球菌PprI蛋白质在毕赤酵母菌中的高效表达及纯化 |
| Efficient expression and purification of Deinococcus radiodurans PprI protein in Pichia pastoris |
| 投稿时间：2015-11-04 |
| DOI：10.3760/cma.j.issn.0254-5098.2016.06.002 |
| 中文关键词: 耐辐射奇球菌 毕赤酵母 PprI蛋白质 纯化 |
| 英文关键词:Deinococcus radiodurans Pichia pastoris PprI protein Purification |
| 基金项目:国家自然科学基金(81372922) |
|
| 摘要点击次数: 2546 |
| 全文下载次数: 2229 |
| 中文摘要: |
| 目的 利用真核毕赤酵母高效表达原核的耐辐射奇球菌pprI基因,建立高效表达及纯化PprI蛋白质的技术路线。方法 根据毕赤酵母密码子的偏爱性,改造耐辐射奇球菌pprI基因的编码序列,利用PCR技术全合成改造过的pprI基因,并在其N末端添加一个6×His标签。PCR产物经纯化后克隆到毕赤酵母表达载体pHBM-905A中,利用Cop I和Not I双酶切并回收线性化的目的片段后,转化毕赤酵母GS115菌株。将获得的毕赤酵母转化子诱导表达,SDS-PAGE、Western blot和质谱检测培养上清液,用Ni-NTA柱纯化目的蛋白,BCA法测定蛋白浓度。结果 新合成的耐辐射奇球菌pprI基因编码序列正确,毕赤酵母转化菌株的培养上清液经SDS-PAGE和Western blot均可检测到相对分子质量为43000的目的蛋白条带,经质谱检测证实该目的蛋白为耐辐射奇球菌PprI蛋白。选用Ni-NTA柱获得了大量纯化的PprI蛋白,应用浓度为250 mmol/L的咪唑淋洗时,PprI蛋白洗脱率最高。BCA法测得纯化蛋白浓度为0.35 mg/ml。结论 建立了一种新的适于毕赤酵母表达的耐辐射奇球菌pprI基因,成功构建了分泌表达PprI蛋白质的重组毕赤酵母工程菌株,建立了高效表达目的蛋白的技术路线,并获得了高效表达和纯化的PprI蛋白。 |
| 英文摘要: |
| Objective To establish a technical route for the efficient expression and purification of PprI protein from Deinococcus radiodurans R1 by using eukaryotic Pichia pastoris. Methods The encoding sequence of the Deinococcus radiodurans pprI gene was modified according to the preference of Pichia pastoris' codon. Modified pprI gene was fully synthesized with PCR and a 6×His tag was added at its N-terminal. The PCR products were purified and then cloned into Pichia pastoris expression vector pHBM-905A. After utilizing Cop I and Not I double enzyme digestion and retrievering linear objective fragment, new pprI gene was transformed to the GS115 strain of Pichia pastoris. The obtained Pichia pastoris transformants were induced to express. Culture supernatants were detected by SDS-PAGE, Western blot, and mass spectrometry. A Ni-NTA column was uesd to purify the target protein and the BCA method was used to determine protein concentration. Results The coding sequence of new synthetic Deinococcus radiodurans pprI gene was correct. The purpose protein band of a molecular weight of 43000 was detected in the culture supernatant of transformed Pichia pastoris strains by SDS-PAGE and Western blot. The mass spectrometry confirmed that it was the Deinococcus radiodurans PprI protein. When the concentration of imidazole was 250 mmol/L, the elution rate of PprI protein was the highest. The purified protein concentration was 0.35 mg/ml measured by BCA method. Conclusions This study has successfully constructed a new pprI gene and the recombinant strain of Pichia pastoris secreting PprI protein, and established a technical route for the efficient expression and purification of PprI protein. |
| HTML 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|