title: 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.
authors.affiliation: Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China.
authors.forename: Jun-Qing
authors.initials: JQ
authors.lastname: Guo
authors.forename: Qing-Mei
authors.initials: QM
authors.lastname: Li
authors.forename: Ji-Yong
authors.initials: JY
authors.lastname: Zhou
authors.forename: Gai-Ping
authors.initials: GP
authors.lastname: Zhang
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Yang
authors.forename: Guang-Xu
authors.initials: GX
authors.lastname: Xing
authors.forename: Dong
authors.initials: D
authors.lastname: Zhao
authors.forename: Shang-You
authors.initials: SY
authors.lastname: You
authors.forename: Chu-Yu
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D017510:Protein Folding; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: C104577:IP10-Mig receptor; D007135:Immunoglobulin Variable Region; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins