Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system

• Published in: Protein expression and purification Vol. 45; no. 1; pp. 168 - 174
• Main Authors: Guo, Jun-Qing, Li, Qing-Mei, Zhou, Ji-Yong, Zhang, Gai-Ping, Yang, Yan-Yan, Xing, Guang-Xu, Zhao, Dong, You, Shang-You, Zhang, Chu-Yu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2006
• Subjects: Animals; Antibody Specificity; Cell Line, Tumor; Cell Movement - genetics; Cell Movement - immunology; Chromatography, Affinity - methods; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Female; Fusion protein; Humans; Immunoglobulin Variable Region - genetics; Inclusion Bodies - chemistry; Inclusion Bodies - genetics; IP10-scFv; Mice; Mice, Inbred BALB C; On-column refolding; Protein Folding; Receptors, Cytokine - chemistry; Receptors, Cytokine - genetics; Receptors, Cytokine - isolation & purification; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; T-Lymphocytes - immunology; Tumor Cells, Cultured; IP10-scFv; Fusion protein; On-column refolding
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.05.016

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 4.48 × 10 −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.