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

  • Hongbo LiEmail author
  • Xiufen Bu
  • Kuai Li
  • Donghai WuEmail author
Original Paper
  • 67 Downloads

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.

Keywords

Poria cocos Immunomodulatory protein Pichia pastoris Purification N-Glycosylation Bioactivity assay 

Notes

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).

Supplementary material

11274_2019_2602_MOESM1_ESM.tif (20 kb)
Supplementary material 1 (TIF 20 KB)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, College of Biological and Food EngineeringHuaihua UniversityHuaihuaChina
  2. 2.Department of Genetics and EugenicsChangsha Hospital for Maternal and Child Health CareChangshaChina
  3. 3.The Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and HealthChinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative MedicineGuangzhouChina

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