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High-level Expression of a Manganese Superoxide Dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris

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Abstract

The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the control of alcohol oxidase 1 promoter with a secretion signal peptide (α-factor) in Pichia pastoris. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting demonstrated that recombinant PoMn-SOD, a 21.8 kDa protein, was secreted into the culture medium. Nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form and the expression system did not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. The maximum enzyme activity (156.9 U/mg) was observed under the following conditions: Initial medium pH was 6.0, induction time point was at the 6th day, and methanol concentration was 0.7 % (v/v). This was the first report on secretory expression of recombinant PoMn-SOD in P. pastoris, which might provide a reference for further practical applications.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31172011) and the Chinese National Science and Technology Support Program (2013BAD16B02). We thank Qin Wang and Jihong Zhu for assistance in conducting Western blot experiment and reviewing the manuscript.

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Authors and Affiliations

  1. Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Chaomin Yin, Wenxia Zhao & Aimin Ma

  2. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Liesheng Zheng & Liguo Chen

  3. National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, 201403, China

    Qi Tan & Xiaodong Shang

Authors
  1. Chaomin Yin
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  2. Wenxia Zhao
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  3. Liesheng Zheng
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  4. Liguo Chen
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  5. Qi Tan
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  6. Xiaodong Shang
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  7. Aimin Ma
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Correspondence to Xiaodong Shang or Aimin Ma.

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Yin, C., Zhao, W., Zheng, L. et al. High-level Expression of a Manganese Superoxide Dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris . Appl Biochem Biotechnol 174, 259–269 (2014). https://doi.org/10.1007/s12010-014-1057-1

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  • DOI: https://doi.org/10.1007/s12010-014-1057-1

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