| Literature DB >> 16125970 |
Jun-Qing Guo1, Qing-Mei Li, Ji-Yong Zhou, Gai-Ping Zhang, Yan-Yan Yang, Guang-Xu Xing, Dong Zhao, Shang-You You, Chu-Yu Zhang.
Abstract
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.Entities:
Mesh：
Substances：
Year: 2005 PMID： 16125970 DOI： 10.1016/j.pep.2005.05.016
Source DB: PubMed Journal: Protein Expr Purif ISSN： 1046-5928 Impact factor: 1.650