Lentiviral Vector-Based Assay System for Quantitative Detection of Intracellular Translocations of Recombinant Proteins

摘要

An enzymatic assay system was developed to quantify the distribution of recombinant proteins over various cell structures. The system takes advantage of α-complementation of □-galactosidase. The large ω fragment of □–galactosidase is expressed in predefined cell structures with the aid of attached protein localization signals. The resulting reporter cell lines are infected with a second construct expressing a target protein fused with the shorter α fragment of □-galactosidase. The physical proximity of the two recombinant proteins carrying the □-galactosidase fragments results in the reconstitution of an active enzyme, and its activity is measured with a plate reader. The recombinant constructs are based on lentiviral vectors and can be rapidly and efficiently introduced into cells by infection with stocks of lentivirus particles. The efficiency of the system was demonstrated with the FOXO3A transcription factor, which shuttles between the cytoplasm and nucleus in the model colon carcinoma cell line RKO.