Expression, purification, and characterization of fusion protein TAT-cytoglobin.
- Author:
Rujing ZHANG
;
Zhaofa LI
;
Weijie SHI
;
Rui'an XU
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Cell Line;
Cell-Penetrating Peptides;
biosynthesis;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
metabolism;
Gene Products, tat;
Genetic Vectors;
Globins;
biosynthesis;
Humans;
Hydrogen Peroxide;
Recombinant Fusion Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2014;30(8):1247-1255
- CountryChina
- Language:Chinese
-
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.
