Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.
A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 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 fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.
Guo
Jun-Qing
JQ
Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China.
Li
Qing-Mei
QM
Zhou
Ji-Yong
JY
Zhang
Gai-Ping
GP
Yang
Yan-Yan
YY
Xing
Guang-Xu
GX
Zhao
Dong
D
You
Shang-You
SY
Zhang
Chu-Yu
CY
D000818:Animals; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D017510:Protein Folding; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
C104577:IP10-Mig receptor; D007135:Immunoglobulin Variable Region; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins