Götz R, Kolbeck R, Lottspeich F, Barde Y A
Max-Planck-Institute for Psychiatry, Department of Neurochemistry and Neurobiochemistry, Martinsried, Federal Republic of Germany.
Eur J Biochem. 1992 Mar 1;204(2):745-9. doi: 10.1111/j.1432-1033.1992.tb16689.x.
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.
神经营养因子-3（NT-3）是一种神经营养因子，最近基于其与神经生长因子（NGF）基因家族其他成员的结构相似性而被克隆出来。为了生产这种蛋白质，构建了一种含有小鼠NT-3编码区的重组痘苗病毒。从感染重组痘苗病毒的细胞收获的条件培养基中含有具有生物活性的NT-3，通过在可控孔径玻璃上的色谱法以及反相和凝胶过滤高压液相色谱法对其进行纯化。从单个细胞工厂培养瓶（1.6升条件培养基）中获得了约200微克纯化的NT-3。N端蛋白质测序表明，成熟因子是在前体的预期位点加工而成的，其氨基酸组成与从DNA序列预测的一致。在从鸡胚制备的解离神经元上测试了重组蛋白的生物活性。使用脊髓感觉神经元，确定使存活率达到半数最大值的纯化重组NT-3浓度为25皮克/毫升。
