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Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
24864 43541 2008 9 PDF Available
Title
Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris
Abstract

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1–243 and 195–418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 μg of full-length rPEDF, and 47 μg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.

Keywords
BHK, baby hamster kidney; ER, endoplasmic reticulum; GFP, green fluorescent protein; GA, golgi-apparatus; GST, glutation S-transferase; HUVEC, human umbilical vein endothelial cells; HPLC, high-performance liquid chromatography; LPLC, low-pressure liquid
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Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 134, Issues 1–2, 20 March 2008, Pages 193–201
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us