河南省农业科学院机构知识库

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

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文献类型：外文期刊

作者： Guo, JQ ¹ ; Li, QM ² ; Zhou, JY ² ; Zhang, GP ² ; Yang, YY ² ; Xing, GX ³ ; Zhao, D; You, SY; Zhang, CY;

作者机构： 1.Zhejiang Univ, Inst Prevent Vet Med, Hangzhou 310029, Peoples R China

2.Zhejiang Univ, Inst Prevent Vet Med, Hangzhou 310029, Peoples R China; Henan Acad Agr Sci, Inst Biotechnol, Zhengzhou 450002, Peoples R China; Wuhan Univ, Coll Life Sci, Wuhan 430072, Peoples R China

3.Zhejiang Univ, Inst Prevent Vet Med, Hangzhou 310029, Peoples R China; Henan Acad Agr Sci, Inst Biotechnol, Zhengzhou 450002, Peoples R China; Wuhan

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 ）

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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.

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