期刊
JOURNAL OF BIOTECHNOLOGY
卷 102, 期 2, 页码 177-189出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0168-1656(03)00020-8
关键词
scFv; acidic isoferritin; inclusion body; His-tag; on-column refolding
A functional single-chain Fv antibody fragment (scFv) specific for acidic isoferritin (AIF) was produced at high level in Escherichia coli. The variable regions of heavy chain (V-H) and light chain (V-L) from the hybridoma 4c9 were connected with a flexible linker using an assembly polymerase chain reaction. The construct Of V-H-linker-V-L was inserted into a phagemid pCANTAB 5 E followed by selection with the Recombinant Phage Antibody System (RPAS). Anti-AIF scFv gene from the recombinant phagemid pCAN4c9 was subcloned into pET28a fused to N-terminal Histag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active anti-AIF scFv was recovered efficiently from inclusion bodies with a refolding yield of approximate 75% confirmed by spectrophotometer. The activity of refolded scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed anti-AIF scFv retains the specific binding activity to AIF with an affinity constant of 7.29 x 10(-8) mol l(-1). The overall yield of anti-AIF scFv with bioactivity in E. coli flask culture was more than 60 mg l(-1). (C) 2003 Elsevier Science B.V. All rights reserved.
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