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 inte rphotoreceptor matrix of the eye, which, although not a proteinase inh ibitor, possesses a number of important biological properties, includi ng promotion of neurite outgrowth and differential expression in quies cent versus senescent states of certain cell types. The low amounts pr esent in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to est ablish a system for overexpression of the recombinant protein for bioc hemical and biological studies. We describe here the expression and se cretion of full-length glycosylated human recombinant PEDF at high lev els (>20 mu g/mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and f luorescence spectroscopies and neurite outgrowth assay. By these assay s, the recombinant protein behaves as expected for a correctly folded full-length human PEDE The availability of milligram amounts of PEDF h as permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards t hermal and guanidine hydrochloride denaturation.