Construction and high-level expression of a single-chain Fv antibody fragment specific for acidic isoferritin in Escherichia coli.
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 His-tag 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).
Guo
Jun-Qing
JQ
College of Life Sciences, Wuhan University, 430072, Wuhan, PR China. whugjq@yahoo.com.cn
You
Shang-You
SY
Li
Le
L
Zhang
You-Zhen
YZ
Huang
Jing-Ning
JN
Zhang
Chu-Yu
CY
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000936:Antigen-Antibody Complex; D014408:Biomarkers, Tumor; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C020391:acidic isoferritin; C055966:immunoglobulin Fv; D005293:Ferritins