Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10843166 | Protein Expression and Purification | 2013 | 7 Pages |
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 20Â mg/L of over 95% highly purified rhPDGF-AA. Mass spectrometry analysis of the purified rhPDGF-AA displayed a molecular weight (MW) of 27,825.513Â Da, composed of a subunit with MW of 15,042.945Â Da and a subunit with MW of 12,904.374Â Da. 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.
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Authors
Hongbo Li, Xiaoyan Hui, Song Yang, Xing Hu, Xiaofeng Tang, Peng Li, Shiwu Li, Lijun Yang, Shouguang Jin, Yu Wang, Aimin Xu, Donghai Wu,