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Title 

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

Authors 

Myung Hoon LeeJu Won Kwak

Publisher 

Elsevier

Issue Date 

2003

Citation 

Journal of Biotechnology, vol. 101, no. 2, pp. 189-198

Keywords 

apolipoprotein B-100Fab′in vitro refoldinginclusion bodymAbB23antigensdialysisDNAescherichia coliplasmid vectors

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.

ISSN 

0168-1656

Link 

http://dx.doi.org/10.1016/S0168-1656(02)00317-6

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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