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Extracellular Production of Human Parathyroid Hormone as a Thioredoxin Fusion Form in Escherichia coli by Chemical Permeabilization Combined with Heat Treatment

Authors

  • Xiang-Yang Fu,

    1. State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China
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  • Wang-Yu Tong,

    Corresponding author
    1. State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China
    • State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Tel: +86–021–64253156. Fax: +86–021–64250068
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  • Dong-Zhi Wei

    Corresponding author
    1. State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China
    • State Key Laboratory of Bioreactor Engineering, Institute of New World Biotechnology, East China University of Science and Technology, Shanghai 200237, China. Tel: +86–021–64253156. Fax: +86–021–64250068
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Abstract

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coliBL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. colitransformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 °C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.

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