Production and characterization of recombinant mouse neurotrophin-3.

Neurotrophin-3 (NT-3) is a neurotrophic-factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 micrograms purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.

[1]  R. Oppenheim.  Cell death during development of the nervous system. , 1991, Annual review of neuroscience.

[2]  L. Reichardt,et al.  Molecular cloning of a human gene that is a member of the nerve growth factor family. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  L. Olson,et al.  Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Y. Kaisho,et al.  Cloning and expression of a cDNA encoding a novel human neurotrophic factor , 1990, FEBS letters.

[5]  D. Goeddel,et al.  Primary structure and biological activity of a novel human neurotrophic factor , 1990, Neuron.

[6]  L. Belluscio,et al.  Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. , 1990, Science.

[7]  Y. Barde,et al.  Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family , 1990, Nature.

[8]  B. Moss.  Regulation of vaccinia virus transcription. , 1990, Annual review of biochemistry.

[9]  K. Eiko,et al.  Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae , 1989 .

[10]  H. Hauser,et al.  Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells. , 1989, European journal of biochemistry.

[11]  H. Thoenen,et al.  Molecular cloning and expression of brain-derived neurotrophic factor , 1989, Nature.

[12]  Yves-Alain Barde,et al.  Trophic factors and neuronal survival , 1989, Neuron.

[13]  K. Neet,et al.  Expression of the cDNA for mouse beta-nerve growth factor protein in Escherichia coli. , 1988, Gene.

[14]  Y. Barde,et al.  Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[15]  W. J. Dower,et al.  High efficiency transformation of E. coli by high voltage electroporation , 1988, Nucleic Acids Res..

[16]  W. Rutter,et al.  Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector , 1988, Molecular and cellular biology.

[17]  B. Moss,et al.  Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors , 1988, Journal of virology.

[18]  H. Stunnenberg,et al.  High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus. , 1988, Nucleic acids research.

[19]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[20]  H. Mewes,et al.  A rapid vapor-phase acid (hydrochloric acid and trifluoroacetic acid) hydrolysis of peptide and protein. , 1987, Journal of biochemistry.

[21]  R. Simpson,et al.  Purification and characterization of human fibroblast-derived hybridoma growth factor identical to T-cell-derived B-cell stimulatory factor-2 (interleukin-6). , 1987, European journal of biochemistry.

[22]  R. Levi‐montalcini.  The nerve growth factor: thirty‐five years later. , 1987, The EMBO journal.

[23]  H. Thoenen,et al.  Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor , 1987, Nature.

[24]  G. Weskamp,et al.  Nerve growth factor gene expression in the developing rat brain. , 1986, Science.

[25]  H. Thoenen,et al.  Placode and neural crest-derived sensory neurons are responsive at early developmental stages to brain-derived neurotrophic factor. , 1985, Developmental biology.

[26]  R. Drillien,et al.  One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[27]  L. Reichardt,et al.  Expression of the beta-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[28]  H. Thoenen,et al.  Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues. , 1984, The EMBO journal.

[29]  P. Fürst,et al.  Measurement of free amino acids in human biological fluids by high-performance liquid chromatography. , 1984, Journal of chromatography.

[30]  B. Moss,et al.  General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes , 1984, Journal of virology.

[31]  H. Thoenen,et al.  Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[32]  H. Thoenen,et al.  Modulation of NGF-induced survival of chick sympathetic neurons by contact with a conditioned medium factor bound to the culture substrate. , 1982, Brain research.

[33]  B. Oakley,et al.  A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. , 1980, Analytical biochemistry.

[34]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[35]  J. Rhim,et al.  Non‐producer human cells induced by murine sarcoma virus , 1975, International journal of cancer.

[36]  R. Angeletti,et al.  Nerve growth factor from mouse submaxillary gland: amino acid sequence. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[37]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[38]  B. Moss.  Inhibition of HeLa Cell Protein Synthesis by the Vaccinia Virion , 1968, Journal of virology.