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Biochemical Characterization of Recombinant Human Nerve Growth Factor
Author(s) -
Schmelzer Charles H.,
Burton Louis E.,
Chan WaiPan,
Martin Evelyn,
Gorman Cori,
CanovaDavis Eleanor,
Ling Victor T.,
Sliwkowski Mary B.,
McCray Glynis,
Briggs Jonathan A.,
Nguyen Tue H.,
Polastri Gian
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb10998.x
Subject(s) - biochemistry , trypsin , peptide sequence , chemistry , gel electrophoresis , amino acid , sodium dodecyl sulfate , chromatography , affinity chromatography , high performance liquid chromatography , recombinant dna , size exclusion chromatography , biology , microbiology and biotechnology , enzyme , gene
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 Arg 119 ‐Ala 120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C‐terminal Arg 118 ‐Arg 119 ‐Ala 120 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.