Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays
| Autor: | Xiufen Bu, Hongbo Li, Donghai Wu, Kuai Li |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Glycosylation Physiology Interleukin-1beta Transfection 01 natural sciences Applied Microbiology and Biotechnology Pichia Pichia pastoris Fungal Proteins Mice 03 medical and health sciences chemistry.chemical_compound N-linked glycosylation Transcription (biology) 010608 biotechnology Complementary DNA Animals Cloning Molecular Sodium dodecyl sulfate Polyacrylamide gel electrophoresis chemistry.chemical_classification 0303 health sciences biology Tumor Necrosis Factor-alpha 030306 microbiology Chemistry General Medicine biology.organism_classification Recombinant Proteins Up-Regulation Reverse transcription polymerase chain reaction RAW 264.7 Cells Biochemistry Protein Multimerization Glycoprotein Wolfiporia Biotechnology |
| Zdroj: | World Journal of Microbiology and Biotechnology. 35 |
| ISSN: | 1573-0972 0959-3993 |
| DOI: | 10.1007/s11274-019-2602-4 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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