Recombinant human pigment epithelium‐derived factor (PEDF): Characterization of PEDF overexpressed and secreted by eukaryotic cells

Recombinant human pigment epithelium‐derived factor (PEDF): Characterization of PEDF... Pigment epithelium‐derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full‐length glycosylated human recombinant PEDF at high levels (>20 μg/mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full‐length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Science Wiley

Recombinant human pigment epithelium‐derived factor (PEDF): Characterization of PEDF overexpressed and secreted by eukaryotic cells

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Publisher
Wiley
Copyright
Copyright © 1996 The Protein Society
ISSN
0961-8368
eISSN
1469-896X
D.O.I.
10.1002/pro.5560051220
Publisher site
See Article on Publisher Site

Abstract

Pigment epithelium‐derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full‐length glycosylated human recombinant PEDF at high levels (>20 μg/mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full‐length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.

Journal

Protein ScienceWiley

Published: Dec 1, 1996

References

  • Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: Antithrombin binding on cultured endothelial cells and perfused rat aorta
    de Agostini, de Agostini; Watkins, Watkins; Slayter, Slayter; Youssoufian, Youssoufian; Rosenberg, Rosenberg

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