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Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays

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Abstract

In this study, the cDNA of immunomodulatory protein from Poria cocos (PCP) was amplified by reverse transcription polymerase chain reaction and used to transform P. Pastoris cells, resulting in rPCP expression as a secreted protein to a concentration of ~ 38 mg/L following methanol induction in shake flasks. Approximately 1.6 mg of high purity rPCP was obtained from a 100-mL culture by Ni+-affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated rPCP as a homologous dimer glycoprotein formed by different molecular-weight monomers. Peptide-N-glycosidase F-mediated deglycosylation analysis showed the presence of an N-glycosylated rPCP monomer, and bioactivity assays showed that rPCP activity upregulated tumor necrosis factor (TNF)-α and interleukin-1β transcription and increased TNF-α secretion from mouse macrophage RAW 264.7 cells. Shortly, we demonstrated successful purification of active rPCP from P. pastoris, which promoted further study of its biological activities and medical applications.

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Acknowledgements

This work was supported by Funds from National Natural Science Foundation of China (81300655) and Scientific Research Fund of Hunan Provincial Education Department (15A147).

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  1. Hongbo Li and Xiufen Bu Parallel first authors.

Authors and Affiliations

  1. Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, College of Biological and Food Engineering, Huaihua University, Huaihua, 418008, China

    Hongbo Li

  2. Department of Genetics and Eugenics, Changsha Hospital for Maternal and Child Health Care, Changsha, 410000, China

    Xiufen Bu

  3. The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, 510530, China

    Kuai Li & Donghai Wu

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  1. Hongbo Li
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  2. Xiufen Bu
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Correspondence to Hongbo Li or Donghai Wu.

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Li, H., Bu, X., Li, K. et al. Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays. World J Microbiol Biotechnol 35, 27 (2019). https://doi.org/10.1007/s11274-019-2602-4

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  • DOI: https://doi.org/10.1007/s11274-019-2602-4

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