HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with S-35-methionine is critica l for the analysis of protein synthesis and turnover. Despite the importanc e of this approach, however, efficient labeling of proteins in vivo is ofte n limited by a low number of available methionine residues, or by deleterio us side-effects associated with protein overexpression. To overcome these l imitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we desc ribe the development of a series of vectors, and corresponding antisera, fo r the expression and detection of HAM-tagged proteins in mammalian cells. W e show that the HAM tag dramatically improves the sensitivity of S-35-methi onine labeling, and permits the analysis of Myc oncoprotein turnover even w hen HAM-tagged Myc is expressed at levels comparable to that of the endogen ous protein. Because of the improved sensitivity provided by the HAM tag, t he vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expre ssion.