Prokaryotic division interactome: setup of an assay for protein-protein interaction mutant selection.
A method which enables selection of protein mutants impaired in their ability to interact with their normal protein partners is presented here. The method is the two-phage two-hybrid assay adapted for mutant selection. In the two-phage assay, the interaction between two proteins enables the formation of a functional hybrid lambdoid repressor that shuts down expression of a reporter gene governed by a chimeric promoter/operator region. To adapt the assay to interaction mutant selection, antibiotic resistance was used as a reporter gene. In this case, the interaction between the two proteins resulted in antibiotic sensitivity, whereas the loss of interaction conferred resistance to the bacterial strain. Therefore, turning on reporter gene expression highlights the loss of interaction due to a mutation in one of the genes for the two protein partners, and leads to direct selection of the mutants regardless of the mutant phenotype. In this paper, application of this method to isolation of interaction mutants in proteins involved in Escherichia coli K12 cytokinesis is reported.
Barbati
S
S
General Microbiology Laboratory, Biology Department, University of Rome, Tor Vergata, Rome, Italy.
Grenga
L
L
Luzi
G
G
Paolozzi
L
L
Ghelardini
P
P
D001435:Bacteriophages / Q000235:genetics; D002678:Chimera; D024881:Drug Resistance, Bacterial; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012641:Selection, Genetic; D020798:Two-Hybrid System Techniques
D029968:Escherichia coli Proteins; D050505:Mutant Proteins