Construction of a tagging system for subcellular localization of proteins encoded by open reading frames

C. H. Chuang, S. C. Hsu, C. L. Hsu, T. C. Hsu, W. J. Syu*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

7 Scopus citations


We have previously characterized a monoclonal antibody (SC1D7) that is directed to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses. To better map the epitope, we took advantage of an available plasmid, pMAL-c2, that encodes the E. coli MBP-coding sequence and constructed plasmids to express MBP fragments. A construct containing the N-terminal portion of MBP does not react with SC1D7, whereas a second construct expressing glutathione S-transferase fused with the C-terminal half of MBP does react with SC1D7. To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7. Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced. This sequence matches MBP with an amino acid stretch of KDPRIAA. To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulting recombinant protein did react with SC1D7. Thereafter, this epitope was incorporated into a eucaryotic expressign plasmid containing a previously defined hepatitis delta virus epitope for protein tagging. This two-epitopetagging vector is useful in various molecular analyses. We demonstrate its usage for localization of a bacterial virulence factor in host cells. This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing.

Original languageEnglish
Pages (from-to)170-175
Number of pages6
JournalJournal of Biomedical Science
Issue number2
StatePublished - 2001
Externally publishedYes


  • Epitope
  • Immunofluorescence
  • Monoclonal antibody
  • Tag


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