Authors: Isarankura-Na-Ayudhya, Chartchalerm
Prachayasittikul, Virapong
Suwanwong, Yaneenart
Tantimavanich, Srisurang
Title: Construction of chimeric antibody binding green fluorescent protein for clinical application
Language (ISO): en
Abstract: A chimeric antibody-binding green fluorescent protein (ZZGFPuv) was successfully constructed and applied as a powerful tool for immunological diagnosis. A gene encoding two repetitive sequences of Z-domain, derivative of IgG-binding B domain of staphylococcal protein A, was fused in-frame to the N-terminus of gfpuv gene. The chimeric gene was subsequently transformed and expressed in various strains of E. coli. Expression of chimeric protein in E. coli strain HB101 resulted in a protein translocation from cytoplasm to periplasmic space and cultivation medium. The chimeric ZZGFPuv could be purified using either IgG Sepharose column or immobilized metal (Cu2+) affinity chromatography. The purified protein migrated in non-denaturing SDS-PAGE as two major bands. A fluorescent band was located at 36 kDa while another band at 48 kDa exhibited non-fluorescence. The fluorescent band was isolated and assessed for IgG-binding via fluorescent emission. The lowest amount of IgG that could be detected by dot immunobinding assay was approximately 630 ng. Indirect immunofluorescent assay for a serological detection of leptospirosis was performed by using the chimeric ZZGFPuv as IgG detector. A strong fluorescent intensity as comparable to that of the fluorescein isothiocyanate (FITC) conjugated system was significantly detected. All these findings support a high feasibility to apply the chimeric Ab-binding GFP for clinical applications in the future.
Subject Headings: Fc binding
green fluorescent protein
Z-domain
URI: http://hdl.handle.net/2003/25652
http://dx.doi.org/10.17877/DE290R-338
Issue Date: 2005-10-07
Appears in Collections:Original Articles

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