Wingfield P, Benedict R, Turcatti G, Allet B, Mermod J J, DeLamarter J, Simona M G, Rose K
Biogen S.A., Geneva, Switzerland.
Biochem J. 1988 Nov 15;256(1):213-8. doi: 10.1042/bj2560213.
Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.
人粒细胞集落刺激因子（G-CSF）及其在第18位残基处由半胱氨酸替换为丝氨酸的突变体在大肠杆菌菌株W3110中产生。从50克湿细胞中获得了约60毫克纯蛋白，回收率约为20%。对这些蛋白进行了物理和化学表征，包括二硫键的测定，发现二硫键存在于第37 - 43位残基和第65 - 75位残基之间。半胱氨酸-18不参与二硫键的形成，用丝氨酸取代后对总体蛋白质构象或生物活性没有影响。野生型和突变体重组衍生蛋白虽然未糖基化，但都具有集落刺激活性。在使用小鼠骨髓单核细胞白血病细胞系WEH1 3B D+的生物测定中，获得的活性与天然G-CSF以及在猴细胞中产生的糖基化重组衍生人G-CSF的活性相似。
