At which end of the E. coli Tagged ORFs are the SPA and TAP tags located?
The SPA and TAP tags were incorporated at the C-terminus.
Zeghouf M et al (2004). Sequential peptide affinity (SPA) system for the identification of mammalian and bacterial protein complexes. Journal of Proteome Research 3, 463–468.
Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism1, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (approx 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data2. This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.