Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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pT7MT, a Metallothionein 2A-Tagged Novel Prokaryotic Fusion Expression Vector

  • Marikar, Faiz M.M.T. (State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University) ;
  • Fang, Lei (State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University) ;
  • Jiang, Shu-Han (State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University) ;
  • Hua, Zi-Chun (State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University)
  • Published : 2007.05.31
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Abstract

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 chromatography, known as $Ni^{2+}$-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 $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/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.

Keywords

References

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