Expression, purification, and characterization of fusion protein TAT-cytoglobin / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 1247-1255, 2014.
Article
in Chinese
| WPRIM
| ID: wpr-345600
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Fusion Proteins
/
Globins
/
Gene Products, tat
/
Cell Line
/
Blotting, Western
/
Electrophoresis, Polyacrylamide Gel
/
Escherichia coli
/
Cell-Penetrating Peptides
/
Genetic Vectors
/
Hydrogen Peroxide
Type of study:
Practice guideline
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2014
Type:
Article
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