Construction of a tagging system for subcellular localization of proteins encoded by open reading frames.
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 expression plasmid containing a previously defined hepatitis delta virus epitope for protein tagging. This two-epitope-tagging 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.
Chuang
C H
CH
Institute of Microbiology and Immunology, National Yang Ming University, Shih-Pai, Taipei 112, Taiwan, ROC.
Hsu
S C
SC
Hsu
C L
CL
Hsu
T C
TC
Syu
W J
WJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D018963:Hepatitis Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003698:Hepatitis Delta Virus; D035923:Hepatitis delta Antigens; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016366:Open Reading Frames / Q000235:genetics*; D021381:Protein Transport; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004274:DNA, Recombinant; D000939:Epitopes; D029968:Escherichia coli Proteins; D018963:Hepatitis Antigens; D035923:Hepatitis delta Antigens; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C442244:hepatitis delta virus large antigen; C461599:maltose transport system, E coli