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| Tat-PTDs Mediated Metallothionein High-yield Expression in Escherichia coli and Its Heavy Metal Resistance Studying |
| LI Hua-ling, WANG Kai, QING Yan, LIU Dan-dan, CHEN Wen-fei |
| Medical College of Yangzhou University, Yangzhou 225001, China |
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Abstract Objective:To explore the feature of Tat-PTDs for metallothionein (MT) high-yield expression in E. coli and its heavy metal resistance in vitro. Methods:the MT-1, 2 encoding genes in Balb/c mice liver tissue was amplified using polymerase chain reaction (PCR) initially, and subcloned into the prokaryotic expression plasmid pET28b-Tat and pET28b followed direct sequencing separately. The validated plasmids were transformed into E. coli BL21 (DE3) cells and induced using 1 mmol/L IPTG, and the protein samples at 0 h, 2 h, 4 h and 6 h were prepared and identified by SDS-PAGE and Western blot. Subsequently, the bacterial cells at 4 h induced were diluted using LB medium and study its resistance of 5 mm copper. Results:This study demonstrated that Tat-PTDs could obviously increase the expression of MT-1, 2 in E. coli and Tat fusion protein mainly expressed in the bacterial supernatant. The Tat fusion protein expression level was 5~8 folds compared to its control according to the results of Western blot and histogram analysis. Furthermore, the over-expressed Tat-MT-1, 2 bacterial cells had an obvious resistance to 5mm copper compared with its control (P<0.01). Conclusion:the data showed that protein transduction based on Tat-PTDs can promote genes of mouse metallothionein high-yield and soluble expression in prokaryotic system. The Tat fusion protein expression level was significantly improved compared to non-Tat fusion protein and its resistance to copper was also increased.
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Received: 20 June 2013
Published: 25 September 2013
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