Document Detail

A vector for double epitope tagging with a recyclable marker.
MedLine Citation:
PMID:  16862609     Owner:  NLM     Status:  MEDLINE    
Multimeric protein complexes play diverse and vital roles in the cell, but following the composition of these complexes under varying growth conditions can be challenging. Toward that goal, we have designed a vector that permits the double epitope tagging of a protein at its carboxy terminus. One 'universal' tag, a triple repeat of the HA1 epitope, is fused with every protein to be studied, allowing the composition and stoichiometry of the proteins in a complex to be detected with a single antibody. Each protein also can be tagged with a second epitope specific for that protein. This 'specific' tag can be used to immunoprecipitate complexes containing that protein of interest. Any epitope to which a specific antibody is available can be used for this second tag. Because there are a limited number of selection markers for cloning in yeast, the kanamycin cassette, flanked by loxP sites, was incorporated into the vector to permit marker recycling using Cre-lox recombinase. This vector was used to tag 4 proteins involved in ribosome biogenesis-Ytm1, Cic1, Brx1 and Drs1. An anti-HA1 antibody could detect all four proteins in crude lysates and yielded the relative abundance of these four proteins, of which Drs1 is reproducibly less abundant than any of the others, which may have implications for the control of ribosome biogenesis. The Ytm1 protein was also tagged with the VSV epitope and can be specifically detected using an anti-VSV antibody. This vector may prove useful for exploring other protein complexes.
Mary Germino; Honeah Sohail; Elizabeth Germino; Joseph Germino
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Yeast (Chichester, England)     Volume:  23     ISSN:  0749-503X     ISO Abbreviation:  Yeast     Publication Date:  2006 Jul 
Date Detail:
Created Date:  2006-07-31     Completed Date:  2006-10-03     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8607637     Medline TA:  Yeast     Country:  England    
Other Details:
Languages:  eng     Pagination:  763-9     Citation Subset:  IM    
Copyright Information:
Copyright (c) 2006 John Wiley & Sons, Ltd.
Department of Internal Medicine, Member, Cancer Institute of New Jersey, University of Dentistry and Medicine, New Jersey, New Brunswick, NJ 08903, USA.
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MeSH Terms
Carrier Proteins / genetics,  metabolism
DNA, Fungal / chemistry,  genetics
Epitopes / genetics*,  metabolism
Genetic Markers
Genetic Vectors / genetics*,  metabolism
Microtubule-Associated Proteins / genetics,  metabolism
Polymerase Chain Reaction
RNA-Binding Proteins / genetics,  metabolism
Saccharomyces cerevisiae / genetics,  metabolism*
Saccharomyces cerevisiae Proteins / genetics*,  metabolism
Reg. No./Substance:
0/BRX1 protein, S cerevisiae; 0/Carrier Proteins; 0/Cic1 protein, S cerevisiae; 0/DNA, Fungal; 0/Epitopes; 0/Genetic Markers; 0/Microtubule-Associated Proteins; 0/RNA-Binding Proteins; 0/Saccharomyces cerevisiae Proteins; 0/Ytm1 protein, S cerevisiae

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