Qiu P, Qin J, Ding Y, Zhu D
Department of Biochemistry, Nanjing University, People's Republic of China.
Biotechnol Appl Biochem. 1995 Feb;21(1):67-75.
Human macrophage colony-stimulating factor (hM-CSF) cDNA joined to the leader region of the precursor of the yeast mating pheromone alpha-factor (MF alpha L) was expressed at high levels in BmN cells and in silkworm (Bombyx mori) larvae, using recombinant Bombyx mori nuclear polyhedrosis virus, as a vector. The biological activity of rhM-CSF detected in the haemolymph was 1 x 10(6) colony-formation units/ml, approximately half of the expression level directed by the native signal peptide of hM-CSF in silkworm larvae. The secreted rhM-CSF was purified to homogeneity. N-terminal analysis showed that the signal peptide had been removed, indicating that insect cells possess the enzymic activity necessary to cleave the pro-alpha-factor leader region from the fusion protein at the carboxy side of Lys-Arg dibasic residues, which is the cleavage site recognized by KEX2 endopeptidase in yeast cells.
将人巨噬细胞集落刺激因子（hM-CSF）的cDNA与酵母交配信息素α因子（MFαL）前体的前导区连接，利用重组家蚕核型多角体病毒作为载体，在BmN细胞和家蚕幼虫中高水平表达。在血淋巴中检测到的重组人巨噬细胞集落刺激因子（rhM-CSF）的生物活性为1×10⁶集落形成单位/毫升，约为家蚕幼虫中hM-CSF天然信号肽指导的表达水平的一半。分泌的rhM-CSF被纯化至同质。N端分析表明信号肽已被去除，这表明昆虫细胞具有在酵母细胞中由KEX2内肽酶识别的切割位点——赖氨酸-精氨酸双碱性残基的羧基侧从融合蛋白上切割前α因子前导区所需的酶活性。
