Song Hai-jing, Zhang Zhuo-mei, Qiu Wei-cheng, Yang Xiao-pan, Wan De-you, He Chun-peng, Li Meng-meng, Gao Xin
Department of Emergency Medicine, PLA 306th Hospital, Beijing, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Sep;28(9):937-9.
To obtain enough human glypican-3 (GPC3) protein for structural and functional research.
The full-length cDNA coding for GPC3 was cloned by RT-PCR from human fetal hepatocytes. The open reading frame (ORF) of the cDNA consists of 1 700 bases, encoding a mature protein of 556 amino acids. The cDNA was inserted into the pPICZ A vector to construct a expression plasmid, named pPICZ A-GPC3. Then the plasmid was transformed into a Pichia pastoris strain, GS115 and the positive strains were screened on the YPD plates with Zeocin. The positive strains were further screened on cellulose acetate and nitrocellulose membrane with HRP labeled His-tag antibody. The selected strains were induced by methanol and the supernatants were analyzed by SDS-PAGE and Western blotting.
SDS-PAGE analysis showed an anticipated band on the gel that could bind with goatanti-GPC3 antibody. Furthermore, the strain was fermented and the expression level was about 5 mg/L, and the recombinant GPC3 protein was purified by cation-exchange chromatography from the fermentation supernatant.
Human GPC3 was expressed successfully in Pichia pastoris and purified to obtain the recombinant protein from fermentation supernatant, which made it possible for further structural and functional studies on GPC3.
获取足够的人磷脂酰肌醇蛋白聚糖-3（GPC3）蛋白用于结构和功能研究。
通过RT-PCR从人胎儿肝细胞中克隆编码GPC3的全长cDNA。该cDNA的开放阅读框（ORF）由1700个碱基组成，编码一个含556个氨基酸的成熟蛋白。将该cDNA插入pPICZ A载体构建表达质粒，命名为pPICZ A-GPC3。然后将该质粒转化入毕赤酵母菌株GS115，在含博来霉素的YPD平板上筛选阳性菌株。用辣根过氧化物酶标记的His标签抗体在醋酸纤维素膜和硝酸纤维素膜上进一步筛选阳性菌株。用甲醇诱导所选菌株，通过SDS-PAGE和Western印迹分析上清液。
SDS-PAGE分析显示凝胶上有一条预期的条带，可与山羊抗GPC3抗体结合。此外，对该菌株进行发酵，表达水平约为5mg/L，通过阳离子交换色谱法从发酵上清液中纯化重组GPC3蛋白。
人GPC3在毕赤酵母中成功表达，并从发酵上清液中纯化得到重组蛋白，这使得对GPC3进行进一步的结构和功能研究成为可能。
