title: [Recombinant expression of mutant human annexin V].
abstract: OBJECTIVE
  To mutate human annexin V gene and transform it to Pichia Pastoris for mutant human annexin V expression, so as to be purified as active annexin V with endogenous metal chelating site.
  
  
  METHODS
  The 5' and 3' end of native annexin V gene were mutated by specific primers. The mutant annexin V gene was inserted into pPIC9K and sequenced. The correct plasmid was linearized and transformed into Pichia Pastoris strain GS115 by electroporation. The transformants were selected from MD plates and cultured in BMGY medium and induced with methanol. The culture was centrifuged and the supernatant was analyzed by SDS-PAGE and silver staining. The binding activity of mutant human annexin V from culture supernatant was determined with phosphatidylserine exposed erythrocytes and fluorescein isothiocyanate-annexin V.
  
  
  RESULTS
  The 5' end of native human annexin V gene was fused with GCAGGCGGCTGCGGCCAT coding sequence and 3' end 946-948 site TGT was mutated to AGC. Pichia Pastoris transformants secreted proteins of relative molecular mass 36 000 48 h after methanol induction. The concentration of this protein that inhibited 50% of the binding of fluorescein-annexin V was 4nmol/L.
  
  
  CONCLUSIONS
  Highly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.
authors.affiliation: Department of Nuclear Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Cai
authors.forename: Fang
authors.initials: F
authors.lastname: L I
authors.forename: Ying-Qiang
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Lian-Fang
authors.initials: LF
authors.lastname: Zheng
authors.forename: Qi
authors.initials: Q
authors.lastname: Pu
mesh_terms: D017304:Annexin A5 / Q000235:genetics*; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D017304:Annexin A5; D012333:RNA, Messenger; D011994:Recombinant Proteins