Takahashi M, Nishida T, Takano M, Yamanishi K, Shimokura M, Ohmoto Y, Aihara K, Ichikawa H, Nakai S, Hirai Y
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Biosci Biotechnol Biochem. 1993 Jun;57(6):915-21. doi: 10.1271/bbb.57.915.
We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200,000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90 kD M-CSF as a homodimer and a molecular form higher than 150 kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42 kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4 x 10(7) units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.
我们通过导入编码554个氨基酸的M-CSF前体和二氢叶酸还原酶（DHFR）基因的表达质粒，并扩增该序列，在中国仓鼠卵巢（CHO）细胞中表达人巨噬细胞集落刺激因子（M-CSF）。获得了一个细胞系，该细胞系在培养6天后分泌约200,000单位/毫升。表达的重组人M-CSF（rhM-CSF）主要由两种分子形式组成，一种主要的80-90kD的M-CSF同二聚体和一种高于150kD的分子形式。纯化主要的rhM-CSF得到一种明显均一的蛋白质，由42kD亚基通过二硫键连接而成，但其中一种纯化的rhM-CSF由分子量为44kD和42kD的两种亚基组成。这些纯化的rhM-CSF具有基本相同的比活性（1至4×10⁷单位/毫克蛋白质）。使用化学方法（三氟甲磺酸）和酶法对后一种rhM-CSF进行去糖基化实验发现，除了N-糖基化和O-糖基化外，还有末端唾液酸，但这两种亚基没有聚合成一个单一分子。
