T7RNA聚合酶的合成与表达+文献综述
关键词 T7启动子 T7 RNA聚合酶 体外转录
毕业设计论文 外文摘要
Title The Tandem Affinity Purification (TAP) Method:A
General Procedure of Protein Complex Purification
Abstract
Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.
Keywords TAP method TAP tag multiple applications
目 次
1 引言 1
1.1 实验背景 1
1.2 T7 RNA 聚合酶简介 2
2 实验原理 5
2.1 构建带有T7启动子及his标签基因的质粒 5
2.2 细胞破碎 6
2.3 Ni柱亲和层析纯化 6
2.4 蛋白质电泳 8
2.5 体外传录 9
2.6 RNA电泳 11
3实验部分 11
3.1 实验流程 11
3.2实验仪器及药品 11
3.3 实验内容 13
4实验结果 18
4.1 T7 RNA 聚合酶的分离提纯 18
4.2 体外转录 19
5 讨论 20
总结 21
致谢 22
参考文献 23
1 引言
1.1 实验背景
随着人类对生物认知的不断深入以及相关技术的快速进步导致的生物研究的不断发展，人们对体外转录得到RNA的要求也越来越强烈。RNA的生物物理和生物化学研究有时需要大量特定长度和特定序列的RNA。DNA/RNA的合成在寡聚核苷酸的合成上有广泛的应用，但是它们很少用于合成长度超过50个碱基的RNA。相反的，T7体外转录系统提供一个简单的方法用于合成纯净、单链的RNA分子，并且这些RNA分子可以是任何长度任何序列。用T7 RNA聚合酶大规模的合成RNA已经广泛地应用于作为生物学活性分子的RNA的研究，比如说酶[1]，转移核糖核酸[2]，以及核糖体RNA[3]。为此相应的T7 RNA聚合酶的应用也不断增加。
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