Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted mush interest as a report er for gene expression. In this;paper, application of capillary electrophor esis with laser-induced fluorescent (CE-LIF) for quantitation of green fluo rescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryo nic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a f actor of six. Detection of a small amount of GFP is difficult by convention al techniques such as fluorescent microscopy due to interference from cell autofluorescence at ow GFP concentrations. The HEK293 cells were transfecte d with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum afte r 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells. (C) 1999 Academic Press.