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Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

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Abstract

Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.

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Acknowledgments

This work was supported by the National High-tech Research and Development Program of China (863 Program, No. 2009AA02Z204), the Major State Basic Research Development Program of China (973 Program; no. 2007CB714306), the National Natural Science Foundation of China (no. 30970057), and the Open Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education (KLIB-KF200902).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu, 214122, China

    Sheng Chen, Zhiguo Liu, Jian Chen & Jing Wu

  2. School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu, 214122, China

    Sheng Chen, Zhiguo Liu, Jian Chen & Jing Wu

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  1. Sheng Chen
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  2. Zhiguo Liu
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  3. Jian Chen
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  4. Jing Wu
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Correspondence to Jian Chen or Jing Wu.

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Chen, S., Liu, Z., Chen, J. et al. Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli . Appl Biochem Biotechnol 165, 666–675 (2011). https://doi.org/10.1007/s12010-011-9286-z

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  • DOI: https://doi.org/10.1007/s12010-011-9286-z

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