pT7MT, a metallothionein 2A-tagged novel prokaryotic fusion expression vector.
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.
State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, PR China.
Faiz M M T
FM
Marikar
Lei
L
Fang
Shu-Han
SH
Jiang
Zi-Chun
ZC
Hua
D017123:Bacteriophage T7 / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D019210:Troponin I / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; C491053:MT2A protein, human; D011993:Recombinant Fusion Proteins; D019210:Troponin I; D008668:Metallothionein; D005982:Glutathione Transferase