Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain.
- Author:
Ai-hua GUO
1
;
Zhi-feng LIU
;
Xue-gang SUN
;
Hai-yu LI
;
Peng DENG
;
Yong JIANG
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Cell Line; Electrophoresis, Polyacrylamide Gel; Genetic Vectors; genetics; Green Fluorescent Proteins; genetics; metabolism; HIV-1; genetics; Humans; Microscopy, Fluorescence; Molecular Sequence Data; Protein Transport; Recombinant Fusion Proteins; biosynthesis; genetics; metabolism; Transfection; methods; tat Gene Products, Human Immunodeficiency Virus; genetics; metabolism
- From: Journal of Southern Medical University 2006;26(5):545-548
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system.
METHODSThe Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency.
RESULTSpET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity.
CONCLUSIONThe prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.
