Abstract
Carboxylesterases are a ubiquitous class of enzymes thought to be involved in xenobiotic metabolism and detoxification. Primary amino acid sequence data suggest that these proteins localize to the endoplasmic reticulum. However, since this family of proteins is highly homologous, the generation of specific reagents to monitor expression and subcellular localization has been unsuccessful. To accomplish in situ detection of a human alveolar macrophage carboxylesterase and a rabbit liver carboxylesterase, we constructed plasmids that expressed recombinant proteins containing an 11 amino acid influenza hemagglutinin tag near the C-terminus. These proteins retained carboxylesterase activity as determined by the conversion of o-nitrophenol acetate to o-nitrophenol. Following transfection of plasmids encoding these proteins into mammalian cells, cells were analyzed by both fluorescence and electron microscopy. The tagged enzymes were localized to the endoplasmic reticulum of both Cos7 monkey kidney cells and Rh30 human rhabdomyosarcoma cells. No tagged protein was detectable in the culture media. Hence, epitope tagging allowed the analysis of expression and localization of specific carboxylesterases. The methods described in this paper are, therefore, applicable to any protein, including those that are highly homologous to other candidate molecules.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
