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

Citation
Ch. Chuang et al., Construction of a tagging system for subcellular localization of proteins encoded by open reading frames, J BIOMED SC, 8(2), 2001, pp. 170-175
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Medical Research General Topics
Journal title
JOURNAL OF BIOMEDICAL SCIENCE
ISSN journal
1021-7770 → ACNP
Volume
8
Issue
2
Year of publication
2001
Pages
170 - 175
Database
ISI
SICI code
1021-7770(200103/04)8:2<170:COATSF>2.0.ZU;2-5
Abstract
We previously characterized a monoclonal antibody body (SC1D7) that is dire cted to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in euca ryotes 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 glutat hione S-transferase fused with the C-terminal half of MBP does react with S C1D7. To precisely define the epitope, random peptides displayed on M13 wer e used to react with SC1D7. Sequences of reactive peptides were aligned, an d a consensus sequence of XDXRIPX was deduced. This sequence matches MBP wi th an amino acid stretch of KDPRIAA. To consolidate the mapping result, a s equence encoding this epitope was inserted into an expression vector and th e resulting recombinant protein did react with SC1D7. Thereafter, this epit ope was incorporated into a eucaryotic expression plasmid containing a prev iously defined hepatitis delta virus epitope for protein tagging. This two- epitope-tagging vector is useful in various molecular analyses. We demonstr ate its usage for localization of a bacterial virulence factor in host cell s. This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing.