蛋白质纯化毕业论文

生物技术专业核心理论课程为植物学、动物学、微生物学、生物化学、分子生物学、细胞生物学、遗传学、生物信息学、基因工程、酶工程、发酵工程、细胞工程。综合实践教学环节包括军训、劳动、思想政治理论课实践、生物学实习、工程训练、生物化学综合大实验、分子生物学综合实验、蛋白质纯化综合大实验、基因工程实验技术、毕业论文（设计）等。

Self-introduction:I am Kong Yuhua, born in Jan 20th, 1984 in JiLin province. I graduated from Dalian Nationalities University with bachelor's degree in July of 2007, and my major is biological engineering. I was born in a harmonious but strict family. My parents teached me to have a sense of responsibility to anything ,which also developed my strict character and desire for the knowledge. I am introverted, enterprising,honest and have stong teamwork spirt, so I can adapt to different condition. I was interested in natural science when I was little, during my study , my biology teacher also influence me , so I choose biological engineering in the college. During the four-years study in the college , I got all-around college basic educatio, I study all the courses hard in biological area with exact goal, I grasped theoretical basis and necessary skill all sidedly. At same time , I put the theory knowledge into practice , when I work as a research assistant, I learnt this experimental concept : Scientific Rigour, this also built up my Practical Ability. After I graduated, I was engaged in Protein Purification in Takara Bio, turned my knowledge into practiceal ability and productive ability .It made me have a deeper understanding to theory and practice of professional knowledge. In work process, I feel the rapid develpment of technology and constantly changing knowledge structure ,which made me have a strong idea for further study. Through further study , master the theory and experiment of bioscience. For this reason, I got an idea to go abroad for graduate study. Korea is the economic power in Asia ,and is the developed country with stable public and friendly people. Secondly, the technology power in your country rank among the world ,is the most ideal campus for further study. Therefore , I want to go to Korea for further study. I sincerely hope that I can go to Korea for study , if it can come true,I think it can expand my horizon ,increase my knowledge and learn advanced professional knowlege ,which can establish a good basis for my future study and work. My major is bioscience ,which include: biochemistry, molecular biology, cytobiology,microbiology, development biology ,genetics and so on. In the period of college , with the carecful guidance of mhy teacher, I learnt the operation of different basic experiments, and learnt cell culture and inspection when I did thesis 5-Fu and the research on CF induce cancer cell apoptosis, I learnt various methods of Protein Purification and related skill of analysis of content and purity. If I can have this opportunity to study in your school, I must grasp this valuable opportunity to require myself strictly to improve professional knowledge,make use of all the time to learn advanced concept . I hope I can have a deeper research in bioscience with the guidance of experts in your school. In order to study and research better, I wil try my best to improve my English ,read extensively all kinds of newest scientific achievements. My goal is to widen my knowledge, enhance Innovative thinking ability, become a professional researcher with both theory and experiment ability. Also put my professional knowledge into practice . Go abroad in Korea , I will put into my total enthusiasm, during this period ,I will try my best to enrich myself , face the future study and life actively. I am so pleased to meet this challenge, I regard it as one of the most steps in my life experiences. For this step, I will get ready for everything, try to make my dream come true.

……………………………………（十） 永远的转录因子第一个模块的实验结束以后，我们进入第二个模块的实验。这个实验的指导教授叫Al Courey。这个模块的实验主要是要从Hela细胞里纯化AP-1。AP1是一种很重要的sequence specific转录因子，在很多生理过程里都有作用。AP-1其实不是一种均一的蛋白质，实际上一种二聚体结构，要么是Jun-Jun 或者是JunFos dimer。Jun和Fos都是alpha helix的结构。Helix的一边有很多Leu,和另一个helix形成Leucine Zipper的结构，而helix另一边就是碱性的氨基酸居多，所以可以和DNA 结合。这个转录因子看起来就象一双筷子夹着DNA。sequence specific转录因子是极其重要的一类蛋白质，真核生物的基因有5~10%是用来编码这种蛋白的。这个模块涉及的一些实验是最经典的一些分离转录因子的方法，所以很有用。怎么才能分离纯化呢？首先要确定要纯化什么因子，换句话说就是要决定纯化跟什么特异DNA序列相结合的转录因子。决定了这个，才能有一个活性检测系统，才能真正开始纯化。另外就是，可以把这种特异序列的DNA固定在柱子上，然后做亲合层析，纯化效率会非常高。整个纯化过程是这样的：首先制备Hela细胞的核提取物，然后用硫酸氨沉淀的办法粗分一下组分，小分子量蛋白(比如Histone H1) 因为无法沉淀而被去掉。到这里比活力会增加到原来的2倍。然后粗分的产物再跑一下凝胶过滤层析，比活力再提高到初始产物的5到10倍。凝胶过滤的产物再过DNA affinity column, 比活力从5-10倍一下子提到了50~100倍。到了这一步，AP-1可以占到蛋白量的 10~50%。值得一提的是这个凝胶过滤层析。我们用的是Sephacryl S-300 HR 凝胶(一种聚丙烯酰胺凝胶)，这种凝胶分辨范围大概是10KDa到几千KDa，但是这种凝胶是比较粗的一种介质，分辨率不能跟superdex之类的比。大概由于不是那么精细，所以可以自己手工装柱，我们用的柱子是Amersham的XK26/40(直径, 高度40cm）。装好的柱子分辨率真的是很低，AP1的洗脱体积跟空体积(void volume）相差挺小，而AP-1只有40~50KD已经很小了。这样一来很多蛋白根本没可能和AP-1分开！这也就是为什么这一步纯化倍数很小的原因。这样一来，为什么要自找麻烦做这又贵又麻烦的一步呢？原因在于这一步可以去掉核提取物里面的核酸酶，所以如果直接跑DNA affinity column, column上的DNA Oligos会被降解掉！另外一个原因是，核提取物有一些可以和DNA非特异结合的蛋白，这些蛋白会饱和DNA affinity column，影响要转录因子的纯化。这个纯化步骤说明，不同的纯化手段必须加以精心组合才会有最好的效果。（十一）核抽提物上回谈到纯化AP-1转录因子的大致流程。其实在过柱子之前的核抽提物的准备也是十分关键的。从天然产物中纯化蛋白，如果大概知道蛋白在什么细胞器官，把细胞器先分离出来作为纯化的起始原料，可以省好多事情。因为细胞器官的分离的方法已经很成熟了。核抽提物是怎么准备的呢？首先，融解Hela细胞。我们在这个模块用的细胞都不是自己长的，而是直接从一个公司Biovest 买来的。据说这个公司有NIH的补贴，所以价钱不贵。一升media的Hela细胞才要17刀。冻存在-80度的hela泡在有glycerol的buffer里面，第一件事情就是要用有镁离子的PBS来冲洗几遍。然后用一种低渗buffer来重悬细胞。这种低渗buffer盐浓度很低，所以由于渗透压的影响，hela细胞膜会变得比较脆弱。这个时候呢，把重悬的细胞用Dounce 匀浆器处理一下,把细胞膜弄破。低渗buffer里头虽然盐浓度很低，但也不是没有盐在里面。主要有两种成分，一个是10mM KCl ,另一个是 MgCl2。KCl没有什么好说的，可以换成NaCl。MgCl2 就关键多了。镁离子可以稳定细胞核, 让细胞核不至于在破碎细胞的时候就破裂。当然说是这么说，一些转录因子还是可能在匀浆的过程就漏出来。细胞膜破碎之后，细胞核还是基本完整的，细胞质里面ER , Golgi之类的东东都漏出来了。然后再来一个低速离心。细胞核是比较重的，所以一离心，马上就沉淀下来了。而其他细胞质或者细胞膜的成分比较轻一些，还留在上清里面。现在想起来，细胞器官的分离，几乎无一例外都是利用细胞器比重不一样来分离的。细胞核是最好分离的了，因为比其他的膜结构重得多。有些细胞器，比如要把Golgi和ER分开，是个十分麻烦的事情，因为比重相差太小了。有些时候，可以用一些古怪的办法来分离。比如lysosome把，比重和线粒体及过氧化酶体很相近。一种办法就是往一堆无辜的耗子注射一种特殊的去垢剂叫Triton WR1339。耗子根本不能分解吸收这种化合物，就积累在肝脏细胞的lysosome里面，使得lysosome比重变轻了一些，这样就可以把lysosome从mitonchondria和peroxysome分出来了。话扯远了，现在再回到AP-1purification。细胞核虽然被沉淀下来了，但里面还有很多染色质,如果DNA都降解漏出来，麻烦就大了。所以下一步就是用高盐buffer( KCl)来破碎细胞核。核膜，染色质之类的垃圾不能溶解，转录因子之类的蛋白却能溶解。再一离心，取上清就行了。我们要的转炉因子就在这里面。按理说，到这一步就可以上柱子了，但是这种上清溶液里面还有不少histone H1，可以抑制体外转录反应。为了祛除hisone, 我们又做了一步硫酸氨沉淀，histone由于太小，沉淀不下来，而转录因子什么的都可以被沉淀下来。沉淀重悬一下就可以跑Sephacryl S-300 的柱子了。