Abstract
Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast α-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 μg/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barnett J, Baecker P, Routledge-Ward C, Bursztyn-Pettegrew H, Chow J, Nguyen B, Bach C, Chan H, Tuszynski MH, Yoshida K, Rubalcava R, Gage FH (1990) Human beta nerve growth factor obtained from a baculovirus expression system has potent in vitro and in vivo neurotrophic activity. Exp Neurol 110:11–24
Bruce G, Heinrich G (1989) Production and characterization of biologically active recombinant human nerve growth factor. Neurobiol Aging 10:89–94
Buxser S, Vroegop S, Decker D, Hinzmann J, Poorman R, Thomsen DR, Stier M, Abraham I, Greenberg BD, Hatzenbuhler NT, Shea M, Curry KA, Tomich C-SC (1991) Single-step purification and biological activity of human nerve growth factor produced from insect cells. J Neurochem 56:1012–1018
Chun LLY, Patterson PH (1977a) Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival growth and differentiation of catecholamine production. J Cell Biol 75:694–704
Chun LLY, Patterson PH (1977b) Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Development study. J Cell Biol 75:705–711
Dicou E, Houlgatte R, Lee J, Wilcken-Bergmann B von (1989) Synthesis of chimeric mouse nerve growth factor precursor and human β-nerve growth factor in Escherichia coli: immunological properties. J Neurosci Res 22:13–19
Edwards RH, Selby MJ, Garcia PD, Rutter WJ (1988a) Processing of the native nerve growth factor precursor to form biologically active nerve growth factor. J Biol Chem 263:6810–6815
Edwards RH, Selby MJ, Mobley WC, Weinrich SL, Hruby DE, Rutter WJ (1988b) Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol Cell Biol 8:2456–2464
Francis PT, Palmer AM, Sims NR, Bowen DM, Davison AN, Esiri MM, Neary D, Snowden JS, Wilcock GK (1985) Neurochemical studies of early-onset Alzheimer's disease: possible influence on treatment. N Engl J Med 313:7–11
Hatanaka H (1981) Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res 22:225–233
Hefti F (1986) Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after fimbrial transection. J Neurosci 6:2155–2162
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kanaya E, Higashizaki T, Ozawa F, Hirai K, Nishizawa M, Tokunaga M, Tsukui H, Hatanaka H, Hishinuma F (1989) Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae. Gene 83:65–74
Kurjan J, Herskowitz I (1982) Structure of a yeast pheromone gene (MFα): a putative α-factor precursor contains four tandem copies of mature α-factor. Cell 30:933–943
Large TH, Bodary SC, Clegg DO, Weskamp G, Otten U, Reichardt LF (1986) Nerve growth factor gene expression in the developing rat brain. Science 234:352–355
Levi-Montalcini R, Angeletti PU (1963) Essential role of the nerve growth factor on the survival and maintenance of dissociated sensory and sympathetic embryonic nerve cells in vitro. Dev Biol 7:653–659
Li AKC, Koroly MJ, Schattenkerk ME, Malt RA, Young M (1980) Nerve growth factor: acceleration of the rate of wound healing in mice. Proc Natl Acad Sci USA 77:4379–4381
Matsuda H, Coughlin MD, Bienenstock JA Denburg JA (1988) Nerve growth factor promotes human hemopoietic colony growth and differentiation. Proc Natl Acad Sci USA 85:6508–6512
Meier R, Becker-Andre M, Gotz R, Heumann R, Shaw A, Thoenen H (1986) Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF. EMBO J 5:1489–1493
Nishizawa M, Ozawa F, Hishinuma F (1989) Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase. Appl Microbiol Biotechnol 32:317–322
Pearce FL, Thompson HL (1986) Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor. J Physiol (Lond) 372:379–393
Perry EK, Tomlinson BE, Blessed G, Bergmann K, Gibson PH, Perry RH (1978) Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. Br Med J 2:1457–1459
Phleps CH, Gage FH, Growdon JH, Hefti S, Harbaugh R, Johnston MV, Khachaturian ZS, Mobley WC, Prince DL, Raskind M, Simpkins J, Thal LJ, Woodcock J (1989) Potential use of NGF to treat Alzheimer's disease. Neurobiol Aging 10:205–207
Sakai A, Ozawa F, Higashizaki T, Shimizu Y, Hishinuma F (1991) Enhanced secretion of nerve growth factor from Saccharomyces cerevisiae using an advanced δ-integration system. Biotechnology 9:1382–1385
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y.
Schechter AL, Bothwell MA (1981) Nerve growth factor receptors on PC12 cells: evidence for two receptor classes with differing cytoskeletal association. Cell 24:867–874
Scott J, Selby M, Urdea M, Quiroga M, Bell GI, Rutter WJ (1983) Isolation and nucleotide sequence of a cDNA encoding the precursor of mouse nerve growth factor. Nature 32:538–540
Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13:8764–8785
Ullrich A, Gray A, Berman C, Dull TJ (1983) Human β-nerve growth factor gene sequence highly homologous to that of mouse. Nature 303:821–825
Waters MG, Evans EA, Blobel G (1988) Prepro-α-factor has a cleavable signal sequence. J Biol Chem 263:6209–6214
Whittemore SR, Ebendal T, Larkfors L, Olson L, Seiger A, Stromberg I, Persson H (1986) Developmental and regional expression of β-nerve growth factor messenger RNA and protein in the rat central nervous system. Proc Natl Acad Sci USA 83:817–821
Whittemore SR, Seiger A (1987) The expression, localization and functional significance of β-nerve growth factor in the central nervous system. Brain Res Rev 12:439–464
Author information
Authors and Affiliations
Additional information
Correspondence to: M. Nishizawa
Rights and permissions
About this article
Cite this article
Nishizawa, M., Ozawa, F., Higashizaki, T. et al. Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae . Appl Microbiol Biotechnol 38, 624–630 (1993). https://doi.org/10.1007/BF00182801
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00182801