Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100

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Title
Expression and functional reconstitution of a recombinant antibody (Fab') specific for human apolipoprotein B-100
Author(s)
Myung Hoon Lee; Ju Won Kwak
Bibliographic Citation
Journal of Biotechnology, vol. 101, no. 2, pp. 189-198
Publication Year
2003
Abstract
We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab′ of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab′ (rFab′) was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab′ had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml-1, whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml-1, where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab′ specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.
Keyword
Apolipoprotein B-100Fab′In vitro refoldingInclusion bodymAbB23
ISSN
0168-1656
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/S0168-1656(02)00317-6
Type
Article
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1. Journal Articles > Journal Articles
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