title: [Construction of eukaryotic plasmid of human GPC3 and expression and purification of recombinant GPC3  protein].
abstract: OBJECTIVE
  To obtain enough human glypican-3 (GPC3) protein for structural and functional research.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Emergency Medicine, PLA 306th Hospital, Beijing, China.
authors.forename: Hai-jing
authors.initials: HJ
authors.lastname: Song
authors.forename: Zhuo-mei
authors.initials: ZM
authors.lastname: Zhang
authors.forename: Wei-cheng
authors.initials: WC
authors.lastname: Qiu
authors.forename: Xiao-pan
authors.initials: XP
authors.lastname: Yang
authors.forename: De-you
authors.initials: DY
authors.lastname: Wan
authors.forename: Chun-peng
authors.initials: CP
authors.lastname: He
authors.forename: Meng-meng
authors.initials: MM
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D005285:Fermentation; D053673:Glypicans / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: C105937:GPC3 protein, human; D053673:Glypicans; D011994:Recombinant Proteins