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Overexpression and Rapid Purification of Rabbit Fast Skeletal Troponin I fromEscherichia coli: Effect of Different Promoters, Host Strains, and Culture Conditions

Protein Expression and Purification
Publication Date
DOI: 10.1006/prep.1994.1083
  • Biology


Abstract Rabbit fast skeletal troponin I (TnI f) cDNA was expressed using two Escherichia coli expression vectors, pRE1 containing the bacteriophage λpL promoter and pAED4, a T7 RNA polymerase-based vector. Although both vectors expressed TnI f, 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 TnI f 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 TnI f 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 TnI f may also be applicable for other eukaryotic proteins.

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