Wingfield P, Graber P, Moonen P, Craig S, Pain R H
Glaxo Institute for Molecular Biology, Genève, Switzerland.
Eur J Biochem. 1988 Apr 5;173(1):65-72. doi: 10.1111/j.1432-1033.1988.tb13967.x.
The conformation and stability of recombinant-derived human and murine granulocyte-macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea-gradient polyacrylamide gel electrophoresis and several spectroscopic methods. The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both alpha-helix and beta-sheet structure. The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding. Comparison of the human E. coli-derived (non-glycosylated) and mammalian cell culture-derived (glycosylated) proteins by urea-gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.
通过分析超速离心、尿素梯度聚丙烯酰胺凝胶电泳以及几种光谱方法，对在大肠杆菌中产生的重组人源和鼠源粒细胞巨噬细胞集落刺激因子的构象和稳定性进行了研究。结果表明，这些蛋白质在物理上是均一的单体蛋白质，具有紧密的球状形状，并且具有相似的二级结构，同时包含α-螺旋和β-折叠结构。两种蛋白质的分子内二硫键对于维持天然构象至关重要，因为用二硫苏糖醇还原会导致蛋白质展开。通过尿素梯度电泳对大肠杆菌来源的（非糖基化）人源蛋白和哺乳动物细胞培养来源的（糖基化）蛋白进行比较，结果表明糖基化对尿素诱导的展开和重折叠的构象稳定性和动力学没有重大影响。
