Schmelzer C H, Burton L E, Chan W P, Martin E, Gorman C, Canova-Davis E, Ling V T, Sliwkowski M B, McCray G, Briggs J A
Genetech, Inc., South San Francisco, California 94080.
J Neurochem. 1992 Nov;59(5):1675-83. doi: 10.1111/j.1471-4159.1992.tb10998.x.
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.
重组人神经生长因子（rhNGF）由中国仓鼠卵巢细胞表达并分泌，通过离子交换和反相（RP）色谱法纯化至同质。通过氨基酸组成、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、RP-HPLC和质谱分析表明，分离得到的产物与120个氨基酸残基的多肽链一致，其N端序列与人类神经生长因子cDNA预期的序列一致。通过尺寸排阻色谱法，rhNGF表现为非共价二聚体。对120个残基单体进行有限的酶切消化产生了另外两种产物，分别为118个残基（胰蛋白酶，去除C端Arg119-Ala120序列）和117个残基（胰蛋白酶加羧肽酶B，去除C端Arg118-Arg119-Ala120序列）。这些产物中的每一种都通过高效离子交换色谱法分离，并通过氨基酸和N端序列分析、SDS-PAGE、RP-HPLC和质谱进行表征。所有三种产物在消化物中均以同二聚体和异二聚体组合形式存在，并且在鸡背根神经节细胞存活和大鼠嗜铬细胞瘤神经突延伸试验中发现具有同等效力。
