Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody.
Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His6-tagged rhA2 was not feasible because of the removal of the N-terminal His6-tag sequence. C-terminally His6-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P. pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.
Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Izumi, Osaka 594-1101, Japan.
Atsushi
A
Tabata
Fumihiko
F
Namba
Minoru
M
Yamada
Taeko
T
Hasegawa
Kumiko
K
Nakahira
Daizo
D
Hamada
Hiroyuki
H
Kitajima
Eiichiro
E
Fukusaki
Itaru
I
Yanagihara
D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006639:Histidine / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D017306:Annexin A2; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D006639:Histidine