Purification and characterization of biologically active recombinant human neurotrophin-3 produced by expression of a chimera gene in Chinese hamster ovary cells.
In order to obtain high-level expression of recombinant human neurotrophin-3 (NT-3), we constructed several types of expression plasmids and examined several cell lines for expression of the human NT-3 gene. The highest level production of the recombinant protein was attained in Chinese hamster ovary cells transfected with an expression plasmid that contains a chimera gene encoding the human nerve growth factor (NGF) prepro-region and human NT-3 mature-region under control of a murine leukemia virus-derived long terminal repeat (MuLV-LTR). This cell line can produce more than 1 mg recombinant human NT-3/1 conditioned medium. The recombinant protein was purified to apparent homogeneity with a cation exchange column, a gel filtration column and a reversed-phase HPLC column with a recovery of about 30%. The purified NT-3, at a concentration as low as 0.2 ng/ml, induced neurite out-growth in neurons prepared from 8-day-old chick embryonic dorsal root ganglia; however, it showed little neurotrophic effect on rat PC12 pheochromocytoma cells, which are known to be NGF-responding cells. In addition, this protein promoted colony formation by human peripheral blood lymphocytes in soft agar culture.
Iwane
M
M
Discovery Research Laboratories, Takeda Chemical Industries Ltd., Osaka, Japan.
Watanabe
T
T
Shintani
A
A
Kaisho
Y
Y
Matsumoto
S
S
Sasada
R
R
Igarashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D020933:Neurotrophin 3; D011498:Protein Precursors / Q000096:biosynthesis / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D017421:Sequence Analysis; D014162:Transfection
D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D011498:Protein Precursors; D011994:Recombinant Proteins