Production, purification and characterization of biologically active recombinant human nerve growth factor

Abstract The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH 2 -terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide likages were determined to be Cys 15 -Cys 80 , Cys 58 -Cys 108 , and Cys 68 -Cys 110 ; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.