Construction of chimeric antibody binding green fluorescent protein for clinical application

Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Suwanwong, Yaneenart; Tantimavanich, Srisurang

Construction of chimeric antibody binding green fluorescent protein for clinical application

dc.contributor.author	Isarankura-Na-Ayudhya, Chartchalerm	de
dc.contributor.author	Prachayasittikul, Virapong	de
dc.contributor.author	Suwanwong, Yaneenart	de
dc.contributor.author	Tantimavanich, Srisurang	de
dc.date.accessioned	2008-06-17T13:58:12Z
dc.date.available	2008-06-17T13:58:12Z
dc.date.issued	2005-10-07	de
dc.description.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.	en
dc.identifier.issn	1611-2156	de
dc.identifier.uri	http://hdl.handle.net/2003/25652
dc.identifier.uri	http://dx.doi.org/10.17877/DE290R-338
dc.language.iso	en	de
dc.relation.ispartofseries	EXCLI Journal ; Vol. 4, 2005	en
dc.subject	Fc binding	en
dc.subject	green fluorescent protein	en
dc.subject	Z-domain	en
dc.subject.ddc	610
dc.title	Construction of chimeric antibody binding green fluorescent protein for clinical application	en
dc.type	Text	de
dc.type.publicationtype	article	de
dcterms.accessRights	open access
eldorado.dnb.zdberstkatid	2132560-1

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