Miyajima A, Otsu K, Schreurs J, Bond M W, Abrams J S, Arai K
EMBO J. 1986 Jun;5(6):1193-7. doi: 10.1002/j.1460-2075.1986.tb04346.x.
Murine (m) and human (h) granulocyte--macrophage colony-stimulating factors (GM-CSF) have been expressed in large quantities in Saccharomyces cerevisiae using a secretion vector containing the promoter and leader sequences of the mating pheromone alpha-factor. Functionally active mGM-CSF was identified by a proliferation assay with a factor-dependent cell line and by a granulocyte--macrophage colony formation assay using bone marrow cells. The activity of hGM-CSF was confirmed by stimulation of granulocyte--macrophage colony formation using human cord blood cells. Murine GM-CSF with various apparent mol. wts (13, 18, 24, 34 and 40 kd, as well as a smear of higher mol. wts) was detected in yeast culture medium by protein blotting using a rat monoclonal antibody specific for the mGM-CSF N-terminal region peptide. Protein blotting using a rat monoclonal antibody specific for the hGM-CSF N-terminal region demonstrated that a 15.6-kd and higher mol. wt heterogeneous species were secreted. Mutations introduced at each of the two potential N-linked glycosylation sites in mGM-CSF showed that the 13-kd protein is not glycosylated and the major 18-kd protein is mainly glycosylated at the more C-terminal site, whereas the heterogeneous higher mol. wt species were not affected by the mutations. The N-terminal amino acid of the 13-kd protein was shown to be Ser which was four amino acids in the C-terminal direction from the fusion point.
利用含有交配信息素α因子启动子和前导序列的分泌载体，已在酿酒酵母中大量表达了小鼠（m）和人（h）粒细胞-巨噬细胞集落刺激因子（GM-CSF）。通过用依赖因子的细胞系进行增殖测定以及使用骨髓细胞进行粒细胞-巨噬细胞集落形成测定，鉴定出具有功能活性的mGM-CSF。通过使用人脐血细胞刺激粒细胞-巨噬细胞集落形成，证实了hGM-CSF的活性。使用对mGM-CSF N端区域肽特异的大鼠单克隆抗体，通过蛋白质印迹法在酵母培养基中检测到具有各种表观分子量（13、18、24、34和40kd，以及更高分子量的条带）的小鼠GM-CSF。使用对hGM-CSF N端区域特异的大鼠单克隆抗体进行蛋白质印迹显示，分泌出一种15.6kd及更高分子量的异质物种。在mGM-CSF的两个潜在N-糖基化位点处引入的突变表明，13kd的蛋白质未被糖基化，主要的18kd蛋白质主要在更靠近C端的位点被糖基化，而异质的更高分子量物种不受这些突变的影响。13kd蛋白质的N端氨基酸显示为丝氨酸，它在融合点C端方向的四个氨基酸处。
