Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo.
A hybrid system which takes advantage of the properties of the lambda repressor allows detection of protein-protein interactions. Fusion of the cI N-terminal domain to a heterologous protein will result in a functional lambda repressor, able to strongly bind to its operator and conferring immunity to lambda infection only when the heterologous protein dimerizes efficiently. In this paper, construction of a recombinant plasmid which allows detection of the activity of the lambda chimeric repressor formed by the N-terminal part of cI fused with a heterologous protein is reported. This construct is interesting due to its potential to be integrated in any target gene of the bacterial host, thus permitting this hybrid assay to be performed, not only in Escherichia coli strains, but in every bacterial genus where the reporter gene can be expressed. In addition, because of its modular construction, this plasmid can be easily modified to be exploitable in many experimental situations, such as in the detection of promoter region activity.
Dipartimento di Biologia, Università 'Tor Vergata' Roma, Via della Ricerca Scientifica, 00133 La Romanina (Roma), Italy.
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G
Di Lallo
Centro Acidi Nucleici del CNR, Roma, Italy.
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P
Ghelardini
Dipartimento di Biologia, Università 'Tor Vergata' Roma, Via della Ricerca Scientifica, 00133 La Romanina (Roma), Italy.
L
L
Paolozzi
D010582:Bacteriophage lambda / Q000235:genetics*; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins