Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.
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 micrograms/ 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.
Stratikos
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E
Department of Biochemistry, University of Illinois-Chicago 60612, USA.
Alberdi
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E
Gettins
P G
PG
Becerra
S P
SP
D000818:Animals; D002942:Circular Dichroism; D006224:Cricetinae; D005136:Eye Proteins; D018014:Gene Transfer Techniques; D006493:Heparin / Q000378:metabolism; D006801:Humans; D009414:Nerve Growth Factors; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D006493:Heparin; D009414:Nerve Growth Factors; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor