Aoki T, Takahashi Y, Koch K S, Leffert H L, Watabe H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan.
FEBS Lett. 1996 Apr 15;384(2):193-7. doi: 10.1016/0014-5793(96)00289-x.
Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.
将水母绿色荧光蛋白（GFP）与蛋白A融合，并在大肠杆菌中表达。荧光天然融合蛋白（PA-GFP）在SDS-PAGE中迁移至47 kDa。然而，非荧光变性PA-GFP迁移至57 kDa，这与理论分子量相符。尽管荧光和非荧光分子之间这种迁移率变化的原因尚不清楚，但天然分子中的小环结构可能会影响它们的迁移率。从产生PA-GFP的大肠杆菌菌株制备的细胞提取物用于蛋白质免疫印迹和斑点印迹。PA-GFP检测的灵敏度和特异性足以进行快速简便的筛选。
