Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system
文献类型: 外文期刊
第一作者: Guo, JQ
作者: Guo, JQ;Li, QM;Zhou, JY;Zhang, GP;Yang, YY;Xing, GX;Zhao, D;You, SY;Zhang, CY
作者机构:
关键词: IP10-scFv;fusion protein;on-column refolding;SINGLE-CHAIN FV;RETAINS ANTIBODY SPECIFICITY;COLONY-STIMULATING FACTOR;ANTITUMOR-ACTIVITY;ESCHERICHIA-COLI;CHEMOKINE;INTERLEUKIN-2;EXPRESSION;RECEPTOR;BINDING
期刊名称:PROTEIN EXPRESSION AND PURIFICATION ( 影响因子:1.65; 五年影响因子:1.548 )
ISSN:
年卷期:
页码:
收录情况: SCI
摘要: 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 pc31P104c9 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 get electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to At F with an affinity constant of 4.48 x 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. (C) 2005 Elsevier Inc. All rights reserved.
分类号: Q51
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