title: High level expression, efficient purification and bioactivity assay of recombinant human platelet-derived growth factor AA dimer (PDGF-AA) from methylotrophic yeast Pichia pastoris.
abstract: Platelet-derived growth factors (PDGFs) are important biochemical mediators regulating many physiological and pathophysiological processes, including promotion of the chemotactic recruitment and proliferation of cells involved in wound repair. Previously, homodimers of rhPDGF-AA protein were purified from Escherichia coli. However, eukaryotic proteins often contain posttranslational modifications, such as glycosylation, that are required for biological functions. In this study, an efficient method was established to purify a glycosylated rhPDGF-AA dimer from P. pastoris culture media by one step CM Sepharose ion exchange chromatography yielding about 20mg/L of over 95% highly purified rhPDGF-AA. Mass spectrometry analysis of the purified rhPDGF-AA displayed a molecular weight (MW) of 27,825.513Da, composed of a subunit with MW of 15,042.945Da and a subunit with MW of 12,904.374Da. The size difference is accounted for by differential glycosylation of the monomers. Biological activity of the rhPDGF-AA was confirmed by its ability to induce NIH/3T3 cells proliferation. The experimental procedure we have developed facilitates production of an active glycosylated rhPDGF-AA in large amounts for further research and drug development.
authors: The Key Laboratory of Regenerative Biology, The Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; The Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Department of Life Sciences, Huaihua College, Huaihua 418008, China.
authors: Hongbo
authors: H
authors: Li
authors: Xiaoyan
authors: X
authors: Hui
authors: Song
authors: S
authors: Yang
authors: Xing
authors: X
authors: Hu
authors: Xiaofeng
authors: X
authors: Tang
authors: Peng
authors: P
authors: Li
authors: Shiwu
authors: S
authors: Li
authors: Lijun
authors: L
authors: Yang
authors: Shouguang
authors: S
authors: Jin
authors: Yu
authors: Y
authors: Wang
authors: Aimin
authors: A
authors: Xu
authors: Donghai
authors: D
authors: Wu
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010843:Pichia / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins; C075278:platelet-derived growth factor A
keywords: Glycosylation; Human platelet-derived growth factor A; Pichia pastoris; Protein purification; Recombinant protein