Production and functional expression of an epitope-tagged human choline acetyltransferase
Résumé
cDNA containing the entire coding region of the human choline acetyltransferase gene (hChAT) was fused to the influenza virus hemagglutinin (HA) epitope preceded by a Kozak sequence. The recombinant HA-hChAT was then inserted into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter. After transient transfection into COS-1 cells, expression was assayed by Northern and Western blot analysis and immunofluorescence. The chimeric HA-hChAT protein was compared to native hChAT for its ability to synthesize acetylcholine. It behaves identically to unmodified hChAT showing that the HA epitope does not affect ChAT activity. This approach enables one to distinguish the expression of the HA-hChAT from endogenous ChAT. Genetically engineered cells that express a high level of HA-hChAT could be used as a promising experimental tool for gene transfer and neurografting techniques as well as to produce and study transgenic mice.