| 李蕊王琛吴放姜清波丁雪张淑平△.蛋白酪氨酸激酶NOK/STYK1 胞内区的表达与纯化*[J].,2014,14(2):201-205 |
| 蛋白酪氨酸激酶NOK/STYK1 胞内区的表达与纯化* |
| Expression and Purification of the Intracellular Domain of Protein TyrosineKinase NOK/STYK1* |
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| DOI： |
| 中文关键词: NOK/STYK1 蛋白表达和纯化 凝胶过滤层析 |
| 英文关键词: NOK/STYK1 Protein expression and purification Gel-filtration chromatography |
| 基金项目:国家自然科学基金项目(81072167 ) |
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| 中文摘要: |
| 摘要目的：酪氨酸蛋白激酶NOK/STYK1 具有很强的促肿瘤形成和转移能力，被认为是很有前途的肿瘤治疗靶点。由于NOK含
有一个跨膜区，且富含疏水性氨基酸，其表达和纯化非常困难，直接影响了对其功能及相关分子机理的深入研究。本研究目的是
获得可溶的且纯度较高的NOK 胞内区融合蛋白△NOK (AA：49-422)，为后续抗体的制备和功能研究奠定重要基础。方法：含有
△NOK基因的原核表达载体，转入BL21 中，IPTG 诱导蛋白表达，通过亲和层析获得可溶的△NOK 融合蛋白。融合蛋白经
凝血酶酶切后，凝胶过滤层析分离标签蛋白获得△NOK蛋白。同时，我们还通过Bac-to-Bac 系统获得含有△NOK基因的杆状病
毒，感染sf9 细胞，尝试在真核细胞中表达目的蛋白。结果：通过在sf9 昆虫细胞和大肠杆菌表达系统中盐浓度等各种条件的摸索，
首次获得了可溶的且纯度较高的NOK 胞内区融合蛋白(△NOK-GST )和一定量去除标签的△NOK 蛋白。本研究中与大肠杆菌相
比，昆虫细胞并不适合△NOK 的纯化。结论：我们建立了一套优化的NOK蛋白表达和纯化体系，从而为后续抗体制备和各种体
内外生化实验等功能研究奠定基础，为研究NOK在肿瘤中的作用和药物筛选创造条件。同时丰富了整个RTKs 家族作用机制的
探索，进一步促进了以RTKs为靶点的治疗手段在临床上的应用。 |
| 英文摘要: |
| ABSTRACT Objective： NOK/STYK1, a novel oncogene with kinase-domain, plays important roles in many kinds of tumors and is considered to be a promising target for cancer therapy. Since it contains a single transmembrane domain and is rich of hydrophobic amino acids, its soluble expression and purification are extremely difficult. Here we tried multiple expression systems to lay an important foundation for its further study, such as preparation of specific antibody and structure analysis. Methods：The recombinant NOK expression plasmid was transformed into the BL21. After induction of IPTG, soluble recombinant protein was lysed and then purified through affinity chromatography. GST tag was cut with thrombin then separated by gel-filtration chromatography. Besides, we attempted to get soluble NOK protein in sf9 cells by infection with recombinant baculovirus which was got through Bac-to-Bac system. Results： The soluble recombinant protein △NOK-GST (AA: 49-422) as well as △NOK without tag was firstly successfully purified by optimization of affecting factors in insect cell and E.coli. Also, it seems that sf9 cells were not suitable for NOK purification compared with E.coli in our research. Conclusion： We established a set of procedures to get soluble recombinant intracellular domain of NOK protein (△NOK-GST) for the preparation of antibodies and in vitro biochemical function studies. This method can be useful for the study on the role of NOK in tumor development and drug screening. Furthermore, this study can extend our understanding on the function mechanisms of RTKs family and promote their clinical applications. |
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