Construction of eukaryotic plasmid of human GPC3 and expression and purification of recombinant GPC3 protein

• Published in: Xi bao yu fen zi mian yi xue za zhi Vol. 28; no. 9; p. 937
• Main Authors: Song, Hai-jing, Zhang, Zhuo-mei, Qiu, Wei-cheng, Yang, Xiao-pan, Wan, De-you, He, Chun-peng, Li, Meng-meng, Gao, Xin
• Format: Journal Article
• Language: Chinese
• Published: China 01.09.2012
• Subjects: Amino Acid Sequence; Fermentation; Glypicans - genetics; Glypicans - isolation & purification; Humans; Molecular Sequence Data; Pichia - genetics; Plasmids; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification
• ISSN: 1007-8738

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 chrom