Overexpression and rapid purification of rabbit fast skeletal troponin I from Escherichia coli: effect of different promoters, host strains, and culture conditions.
Rabbit fast skeletal troponin I (TnIf) cDNA was expressed using two Escherichia coli expression vectors, pRE1 containing the bacteriophage lambda pL promoter and pAED4, a T7 RNA polymerase-based vector. Although both vectors expressed TnIf, overexpression of the target protein was achieved with pAED4. The effect of several parameters such as culture condition, compatible host strain, and inhibition of protein synthesis by rifampicin on the expression of TnIf was investigated. The overexpressed target protein synthesized during a brief induction period of only 2 h was conveniently purified from inclusion bodies by a simple and rapid procedure involving extraction with urea, ultracentrifugation, DE-52 column chromatography, and gel filtration. About 50-75 mg of highly purified TnIf was obtained per liter E. coli culture by this method, which does not involve time-consuming multistep procedures such as affinity and ion exchange chromatography as previously reported in the literature. The isolated unfused protein is stable and is indistinguishable from native protein in all biological parameters examined. The parameters optimized in this report for overexpression of TnIf may also be applicable for other eukaryotic proteins.
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Department of Anatomy and Cellular Biology, School of Veterinary Medicine, Tufts University, Boston, Massachusetts 02111.
Du
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Wu
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Leavis
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Sarkar
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D002479:Inclusion Bodies; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012293:Rifampin; D014336:Troponin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D003470:Culture Media; D014336:Troponin; D000251:Adenosine Triphosphatases; D012293:Rifampin