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

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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Guo JQ,Li QM,Zhou JY,Zhang GP,Yang YY,Xing GX,Zhao D,You SY,Zhang CY

doi

10.1016/j.pep.2005.05.016

keywords:

subject

Has Abstract

pub_date

2006-01-01 00:00:00

pages

168-74

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00176-2

journal_volume

45

pub_type

杂志文章

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