Production, purification and characterization of biologically active recombinant human nerve growth factor.
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 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-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 linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; 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.
Iwane
M
M
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Kitamura
Y
Y
Kaisho
Y
Y
Yoshimura
K
K
Shintani
A
A
Sasada
R
R
Nakagawa
S
S
Kawahara
K
K
Nakahama
K
K
Kakinuma
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000235:genetics / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis; D011994:Recombinant Proteins
D009414:Nerve Growth Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins