Hua Z, Liang X, Zhu D
Department of Biochemistry, Nanjing University, P.R. China.
Biochem Mol Biol Int. 1994 Sep;34(2):419-27.
A truncated human macrophage colony stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was obtained by using polymerase chain reaction. When inserted into plasmid pCXJ1 and psPHO5 and introduced into Kluyveromyces lactis, it directs the the secretory expression of the biologically active dimeric form of M-CSF. Through a four-step purification protocol, i.e. ammonium sulfate salting out, DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified to homogenerity and show its apparent molecular mass at 21KDa on reduced SDS-PAGE, with a specific activity of 1.21 x 10(7) units/mg protein.
通过聚合酶链反应获得了截短的人巨噬细胞集落刺激因子（M-CSF）cDNA，其编码天然M-CSF的3至149个氨基酸残基。当将其插入质粒pCXJ1和psPHO5并导入乳酸克鲁维酵母中时，它指导M-CSF生物活性二聚体形式的分泌表达。通过硫酸铵盐析、DEAE-纤维素柱色谱、苯基琼脂糖疏水色谱和Mono Q快速蛋白质液相色谱这四步纯化方案，重组截短的M-CSF被纯化至均一，在还原SDS-PAGE上显示其表观分子量为21KDa，比活性为1.21×10⁷单位/毫克蛋白质。
