Biochemical characterization of recombinant human nerve growth factor.
Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.
Schmelzer
C H
CH
Genetech, Inc., South San Francisco, California 94080.
Burton
L E
LE
Chan
W P
WP
Martin
E
E
Gorman
C
C
Canova-Davis
E
E
Ling
V T
VT
Sliwkowski
M B
MB
McCray
G
G
Briggs
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014162:Transfection
D009414:Nerve Growth Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins