Wang Y, Wang G, O'Kane D J, Szalay A A
Department of Biochemistry, Loma Linda University, CA 92350, USA.
Mol Gen Genet. 2001 Jan;264(5):578-87. doi: 10.1007/s004380000322.
We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.
我们之前曾报道，含有由海肾荧光素酶与水母绿色荧光蛋白（GFP）组成的融合蛋白的大肠杆菌和哺乳动物细胞，在腔肠素存在的情况下表现出从荧光素酶到GFP的发光共振能量转移（LRET）。在本文中，我们描述了两种基因融合体的构建，其中胰岛素样生长因子II（IGF-II）的cDNA与“人源化”GFP的cDNA相连，胰岛素样生长因子结合蛋白6（IGFBP-6）的cDNA与编码海肾荧光素酶（RUC）的cDNA相连。基于使用抗GFP和海肾荧光素酶的抗体，融合基因构建体在CHO细胞中的表达分别产生了预期分子量的单条多肽，分别对应IGF-II-GFP和IGFBP-6-RUC。通过荧光显微镜验证了CHO细胞中IGF-II-GFP的分泌，并通过发光测定法确认了培养基中存在IGFBP-6-RUC。在腔肠素存在的情况下，使用低光成像系统和荧光分光光度法，通过测量从IGFBP-6-RUC蛋白到IGF-II-GFP的LRET来监测两种已知结合伴侣IGF-II和IGFBP-6之间的相互作用。基于这些数据，荧光素酶到GFP的LRET在实时研究真核细胞中的蛋白质-蛋白质相互作用方面具有很大的前景。
