肿瘤转移关键酶:人乙酰肝素酶氨基端亚基的表达纯化及鉴定  

Expression,purification and characterization of human heparanase N-terminal subunit protein

作　　者：李燕杰 李吉学 凌焱[2] 汤国营[2] 宋子博[3] 林艳丽[2] 朱厚础[2] 

机构地区：[1]郑州大学医学实验中心癌症研究室,郑州450052 [2]军事医学科学院生物工程研究所,北京100071 [3]河南省人民医院医学遗传研究所,郑州450003

出　　处：《军事医学科学院院刊》2005年第3期236-240,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:乙酰肝素酶在肿瘤转移的过程中起重要作用,其氨基端小亚基蛋白(Mr为8×103)对其功能活性是不可缺少的。得到此蛋白将有助于深入研究乙酰肝素酶的功能活性。方法:我们从人胎盘cDNA文库中分离乙酰肝素酶蛋白全长编码基因,将乙酰肝素酶氨基端小亚基的基因克隆入pGE-X2TK,转化大肠杆菌进行融合表达。经发酵,纯化,复性得到乙酰肝素酶的氨基端亚基蛋白。结果:得到6.9g纯度>90%的氨基端小亚基融合蛋白。GSTpull-down实验表明,此融合蛋白可与乙酰肝素酶羧基端大亚基蛋白结合。复性后的融合蛋白可被激酶切割为GST蛋白和乙酰肝素酶氨基端亚基蛋白。结论:所得的乙酰肝素酶的氨基端小亚基蛋白为进一步研究该亚基蛋白在细胞内的免疫亚定位及功能奠定了基础。Objectives:To express and purify the N-terminal subunit of haparanase which plays an important role in metastasis. Methods: The full encoding gene was isolated from the human placenta cDNA library and the N-terminal subunit gene was cloned into the pGEX-2TK vector for expressing the N-terminal subunit protein. After the fermentation, a series of purifications and renaturing,the target protein got weighted (6.9 g) and the purity was about 90%. Results: GST pull down assay presented that the expressed fusion protein could combine with the C-terminal subunit protein of heparanase. The target fusion protein could be cleaved into GST protein and the heparanase N-terminal subunit protein. Conclusion: The N-terminal subunit protein of heparanase obtained can be used for studying the immuno-sublocation and function of the N-terminal subunit.

关 键 词：乙酰肝素酶 氨基端亚基蛋白 融合表达 纯化 肿瘤转移 

分 类 号：R73-3[医药卫生—肿瘤]