Investigation into the use of C‐ and N‐terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays

Abstract

Abstract Reverse transfection microarrays were described recently as a high throughput method for studying gene function. We have investigated the use of this technology for determining the subcellular localization of proteins. Genes encoding 16 proteins with a variety of functions were placed in Gateway expression constructs with 3′ or 5′ green fluorescent protein (GFP) tags. These were then packaged in transfection reagent and spotted robotically onto a glass slide to form a reverse transfection array. HEK293T cells were grown over the surface of the array until confluent and GFP fluorescence visualized by confocal microscopy. All C‐terminal fusion proteins localized to cellular compartments in accordance with previous studies and/or bioinformatic predictions. However, less than half of the N‐terminal fusion proteins localized correctly. Of those that were not in concordance with the C‐terminal tagged proteins, half did not exhibit expression and the remainder had differing subcellular localizations to the C‐terminal fusion protein. This data indicates that N‐terminal tagging with GFP adversely affects the protein localization in reverse transfection assays, whereas tagging with GFP at the C‐terminal is generally better in preserving the localization of the native protein. We discuss these results in the context of developing high‐throughput subcellular localization assays based on the reverse transfection array technology. Copyright © 2004 John Wiley & Sons, Ltd.