Gillis S, Urdal D L, Clevenger W, Klinke R, Sassenfeld H, Price V, Cosman D
Department of Molecular Biology, Immunex Corporation, Seattle, Washington 98101.
Behring Inst Mitt. 1988 Aug(83):1-7.
Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.
已经建立了用于生产重组人粒细胞巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-3（IL-3）和粒细胞集落刺激因子（G-CSF）的高效酵母表达和纯化系统。尽管通过酵母生产重组集落刺激因子（通过基于分泌的系统）能够产生天然分子，然后可以很容易地从发酵液中分离出来，但在许多情况下，天然cDNA必须进行改造以实现高效表达，并生产出异质性较低的产品。对于本文所述的集落刺激因子，有益的突变（通过定点诱变产生）包括消除潜在的N-连接糖基化位点、去除KexII蛋白酶识别位点（特别是双碱性序列中的改变）以及消除可能因分子间二硫键而使均一产品分离复杂化的多余半胱氨酸残基。
