| Autor: |
Marikar FM; State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, PR China., Fang L, Jiang SH, Hua ZC |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of microbiology and biotechnology [J Microbiol Biotechnol] 2007 May; Vol. 17 (5), pp. 728-32. |
| Abstrakt: |
In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT (GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A (MT2A). There are in-framed multiple cloning sites (MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame (ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatogralhy, known as Ni2+-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step Ni2+ -affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30 mg/l and 28 mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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