Expression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cells.
Antibody fragments and their fusion proteins are indispensable tools as immunoassay reagents in diagnostics and molecular/cellular biotechnology. However, bacterial expression of cloned antibody genes with correct tertiary structure is not always guaranteed because of the lack of proper folding machinery and/or post-translational modifications. In addition, frequently used bacterial alkaline phosphatase as a fusion partner generally shows lower specific activity than the mammalian enzyme, which hampers its wider use as a detection reagent. Here we tried to express the fusion proteins of antibody variable region(s) and secreted human placental alkaline phosphatase (SEAP) using mammalian cell culture. As a result, functional V(H)-SEAP and single-chain Fv-SEAP fusion proteins were successfully obtained from COS-1 cells, which was confirmed by ELISA and Western blotting. This system will be applicable to the rapid production of various antibody-enzyme fusions suitable for ELISA and open-sandwich ELISA that utilizes antigen-dependent V(H)/V(L) interaction for antigen quantitation.
Sasajima
Yoshiyuki
Y
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Iwasaki
Ryohei
R
Tsumoto
Kouhei
K
Kumagai
Izumi
I
Ihara
Masaki
M
Ueda
Hiroshi
H
D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D014162:Transfection
D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase