[Recombinant expression of mutant human annexin V].
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.
Cai
Jiong
J
Department of Nuclear Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
L I
Fang
F
Zhang
Ying-Qiang
YQ
Zheng
Lian-Fang
LF
Pu
Qi
Q
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
D017304:Annexin A5; D012333:RNA, Messenger; D011994:Recombinant Proteins