Expression and purification of recombinant human zona pellucida proteins
Expression and purification of recombinant human zona pellucida proteins
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DOI：
10.1006/prep.1999.1060
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发表时间：
1999-07-01
影响因子：
1.6
通讯作者：
Liu, HF
中科院分区：
文献类型：
--
作者：
Harris, JD;Seid, CA;Liu, HF
重组人透明质酸（rhZP）蛋白（减去N-末端前导序列和C-末端跨膜样结构域）在四种不同的表达系统中表达：细菌、酵母、昆虫细胞和中国仓鼠卵巢（CHO）细胞。每个系统中的重组蛋白用C-末端六个组氨酸（His 6）区段进行工程化，所述区段用于通过金属亲和[镍（Ni）或钴（Co）]柱色谱法纯化蛋白。每种rhZP蛋白都是作为免疫避孕疫苗的候选抗原。然而，在细菌、酵母和昆虫细胞培养物中产生的rhZP蛋白必须用强变性剂溶解后才能纯化。纯化后，这些表达系统中的每一个的最终产物需要6 M尿素以保持溶解度。而CHO细胞表达的rhZP蛋白可分泌到培养基中，且可溶性蛋白可纯化至接近均一。在本报告中，表达和纯化程序用于生产和分离这些分泌的蛋白质进行了描述。(C)北京：科学出版社.
Recombinant human zona pellucida (rhZP) proteins (minus the N-terminal leader and the C-terminal transmembrane-like domain) were expressed in four different expression systems: bacteria, yeast, insect cells, and Chinese Hamster Ovary (CHO) cells. The recombinant proteins in each system were engineered with a C-terminal six histidine (His6) segment that was used to purify the proteins by metal affinity [either nickel (Ni) or cobalt (Co)] column chromatography. Each of the rhZP proteins was a candidate antigen as an immunocontraceptive vaccine. However, the rhZP proteins produced in bacteria, yeast and insect cell culture could only be purified after being solubilized by strong denaturants. After purification the final products of each of these expression systems required 6 M urea to maintain solubility. However, the rhZP proteins expressed by CHO cells were secreted into the media, and the soluble proteins could be purified to near homogeneity. In this report the expression and purification procedures used to produce and isolate these secreted proteins are described. (C) 1999 Academic Press.