Joitrnal zyxwvutsrqponm of Neurochemistry zyxwvutsrqponm Raven Press, Ltd., zyxwvutsrqpon New York zyxwvutsrqp 0 1992 International Society for Neurochemistry Biochemical Characterization of Recombinant Human Nerve Growth Factor Charles H. Schmelzer, Louis E. Burton, Wai-Pan Chan, Evelyn Martin, Cori Gorman, Eleanor Canova-Davis, Victor T. Ling, Mary B. Sliwkowski, Glynis McCray, Jonathan A. Briggs, Tue H. Nguyen, and Gian Polastri zyx Genentech, Inc., South Sun Francisco, California, U.S.A. Abstract: 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, so- dium 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 1 18 (trypsin, removal of the C-terminal Arg"9-Ala'Zo sequence) and 1 17 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg"'- Arg"9-Ala'20 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatogra- phy and characterized by amino acid and N-terminal se- quence analyses, SDS-PAGE. RP-HPLC, and mass spec- trometry. 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 gan- glion cell survival and rat pheochromocytoma neurite ex- tension assays. Key Words: Nerve growth factor-Purifica- tion-Bioequivalence-Dimers. Schmelzer C. H. et al. Bio- chemical characterization of recombinant human nerve growth factor. J. Neurochem. 59, 1675-1683 (1992). Nerve growth factor (NGF) is a protein required for the growth and survival of sympathetic and sensory neurons during development and in mature animals (Thoenen and Barde, 1980; Yankner and Shooter, jor source of this protein for biochemical and biologi- cal study has been the mouse submaxillary gland (Co- hen, 1960; Varon et al., 1967a), although other tissue sources have been examined, most notably snake ven- oms (Hogue-Angeletti and Bradshaw, 1979)and pros- tate gland and its secretions (Harper et al., 1979, 1982; Harper and Thoenen, 1980; Chapman et al., 198 1). The mouse submaxillary gland source yields two primary types of the active factor, dependent on 1982; L~i-M~fitdd~~i, 1987). zyxwvut LT~ltil t.~~fifly, the ma- the approach to purification: the 7s NGF complex (Varon et al., 1967a,b, 1968; Smith et al., 1968) and 2.5S, or P-NGF (Bocchini and Angeletti, 1969). p- NGF, the subunit of the 7s NGF complex exhibiting NGF activity, is a noncovalent dimer of two zy - 13- kDa polypeptides, the monomer of which occurs in one of three commonly described forms: 118 residues, or 6-NGF; 1 17 residues, or des Arg'18 P-NGF; and 1 10 residues, or des octa (or des 1-8) P-NGF (Moore et al., 1974; Mobley et al., 1976). Each of these forms has been well characterized, and their presence in mouse submaxillary gland preparations is also depen- dent on the mode of isolation (for review, see Longo et al., 1989). Received December 3 1, 199 1; revised manuscript received April 6, 1992; accepted April 14, 1992. Address correspondence and reprint requests to Dr. C. H. Schmelzer at Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, U.S.A. Abbreviations used: CF, culture fluid; CHO, Chinese hamster ovary; DEFF, diethylaminoethane-Sepharose fast flow; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked im- munosorbent assay; HPIEC, high-performanceion-exchange chro- matography; MOPSO, 3-(N-morpholino)-2-hydroxypropanesul- fonic acid; NGF, nerve growth factor; rhNGF, recombinant human nerve growth factor; RP, reversed-phase; SDS-PAGE, sodium do- decyl sulfate-polyacrylamide gel electrophoresis; SEC, size-exclu- sion chromatography; SSFF. sulfopropyl-Sepharose fast flow; TFA, trifluoroacetic acid. Unless otherwise indicated, the abbreviation for the form of the NGF molecule represents the homodimeric form. In the case of heterodimers, the different monomers in the dimeric complex are separated by a colon (:). For example, 120: 1 18 represents the heterodimer between the two rhNGF monomers of 120 and 1 18 residues. 1675