In order to perform large-scale analysis of protein complexes in Escherichia coli
, researchers at the University of Toronto have created a library of affinity-tagged open reading frames. With c-terminal tandem affinity purification (TAP) or sequential peptide affinity (SPA) tagged alleles of over 800 ORFs, the E. coli
Tagged ORF library allows purification of tagged proteins and their interacting protein partners.
To create this library, ORFs were selected to represent broad biological coverage, including highly-conserved essential and non-essential proteins, proteins with putative functional assignments, and hypothetical uncharacterized ORFs. Then, using a phage recombination system, sequence-specific PCR products encoding affinity purification tags were inserted into discrete loci of the E. coli chromosome. 857 proteins, including 198 essential and conserved proteins, were successfully tagged. Over a quarter of these proteins were also successfully purified by the source lab. Proteins were isolated by affinity purification and their binding partners identified using LC-MS and MALDI-TOF mass spectrometry.
Generate a reliable network of functionally diverse protein complexes
Gain insight into the function of uncharacterized proteins
Outline the topological organization of the bacterial interactome
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