Refolding and characterization of human recombinant heparin-binding neurite-promoting factor.
Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)
Seddon
A P
AP
Department of Protein Chemistry, American Cyanamid Company, Lederle Laboratories, Pearl River, New York 10965.
Hulmes
J D
JD
Decker
M M
MM
Kovesdi
I
I
Fairhurst
J L
JL
Backer
J
J
Dougher-Vermazen
M
M
Böhlen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D003553:Cystine / Q000096:biosynthesis; D016207:Cytokines / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli; D005346:Fibroblast Growth Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008647:Mesocricetus; D000078792:Midkine; D008969:Molecular Sequence Data; D017510:Protein Folding; D017468:Receptors, Fibroblast Growth Factor / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D016207:Cytokines; D018076:DNA, Complementary; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; C066845:pleiotrophin; D000078792:Midkine; D003553:Cystine; D005346:Fibroblast Growth Factors; D003545:Cysteine; D004229:Dithiothreitol