title: [Expression, purification, and characterization of fusion protein TAT-cytoglobin].
abstract: he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.
authors.forename: Rujing
authors.initials: R
authors.lastname: Zhang
authors.forename: Zhaofa
authors.initials: Z
authors.lastname: Li
authors.forename: Weijie
authors.initials: W
authors.lastname: Shi
authors.forename: Rui'an
authors.initials: R
authors.lastname: Xu
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis*; D000078842:Cytoglobin; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015696:Gene Products, tat; D005822:Genetic Vectors; D005914:Globins / Q000096:biosynthesis*; D006801:Humans; D006861:Hydrogen Peroxide; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: C000627265:CYGB protein, human; D057846:Cell-Penetrating Peptides; D000078842:Cytoglobin; D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D005914:Globins; D006861:Hydrogen Peroxide