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The construction of a bifunctional fusion protein consisting of SEC2 and EGFP
Author(s) -
Liu Yanli,
Xu Mingkai,
Li Xu,
Sun Jian,
Zhang Chenggang,
Zhang Huiwen
Publication year - 2014
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1203
Subject(s) - green fluorescent protein , fusion protein , protein subcellular localization prediction , flow cytometry , microbiology and biotechnology , chemistry , fluorescence microscope , plasmid , recombinant dna , biology , fluorescence , biochemistry , gene , physics , quantum mechanics
The aim of this study was to construct a bifunctional fusion protein consisting of staphylococcal enterotoxin C2 ( SEC2 ) and enhanced green fluorescent protein ( EGFP ). We inserted EGFP and SEC2 fragments into the pET‐28a (+) vector to create the expression plasmid vector, pET‐28a(+)‐SEC2‐EGFP , using a two‐step method. After verification of the plasmid, successful isolation of the fusion protein, SEC2‐EGFP , was achieved by Ni + ‐affinity chromatography. Fluorescence microscopy, methylthiazol tetrazolium, and flow cytometry assays demonstrated that the constructed fusion protein not only retained the fluorescence signal of EGFP but also exhibited SEC2 bioactivity. Therefore, SEC2‐EGFP is a promising tool for the study of the detailed temporal and spatial distributions of SEC2 in cells. Future studies with this vector may help uncover novel therapeutic strategies to treat or manage SEC2 ‐associated diseases and be a new clinical tool for exploiting SEC2 in immunotherapy.