Abstract
Epitope-tagging of proteins has become a widespread technique for the analysis of protein function, protein interactions and protein localization among others. Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo. Different systems have been developed during years in the yeast Saccharomyces cerevisiae. In the present study, we analysed systematically a set of yeast proteins that were fused to different tags. Analysis of the tagged proteins revealed an unexpected general effect on protein level when some specific tagging module was used. This was due in all cases to a destabilization of the proteins and caused a reduced protein activity in the cell that was only apparent in particular conditions. Therefore, an extremely cautious approach is required when using this strategy.
Highlights
Immunological methods have been revealed as crucial in the development of molecular and cellular biology
green fluorescent protein (GFP) tag is an invaluable tool for localizing proteins in both living and fixed cells [5,6,7], and both the HA epitope derived from influenza virus hemagglutinin [8], and the myc epitope derived from the mammalian cmyc protein [9], have been widely used for protein localization by immunofluorescence and for biochemical detection and isolation of proteins
The fact that this effect was detected in all six different proteins investigated, and that the same results were observed using a different yeast background, strongly suggests that the observed reduction in protein level after C-terminal 3xHA tagging is not an artefact caused by altered antigen-antibody recognition but a genuine effect in protein level caused by the tagging
Summary
Immunological methods have been revealed as crucial in the development of molecular and cellular biology. Fusion-tag technology has grown-up to facilitate protein analysis, purification and visualization. Fusion tags relevant for this manuscript can vary greatly in size, from large tags such as the Aequorea victoria green fluorescent protein (GFP) with a size of 26,9 kDa, to small peptide tags including multiple copies of c-myc and HA, each around 1 kDa. GFP tag is an invaluable tool for localizing proteins in both living and fixed cells [5,6,7], and both the HA epitope derived from influenza virus hemagglutinin [8], and the myc epitope derived from the mammalian cmyc protein [9], have been widely used for protein localization by immunofluorescence and for biochemical detection and isolation of proteins
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