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Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo

¹ Dipartimento di Biologia, Università ‘Tor Vergata’ Roma, Via della Ricerca Scientifica, 00133 La Romanina (Roma), Italy
² Centro Acidi Nucleici del CNR, Roma, Italy

ABSTRACT

A hybrid system which takes advantage of the properties of the repressor allows detection of protein-protein interactions. Fusion of the cl N-terminal domain to a heterologous protein will result in a functional repressor, able to strongly bind to its operator and conferring immunity to infection only when the heterologous protein dimerizes efficiently. In this paper, construction of a recombinant plasmid which allows detection of the activity of the chimeric repressor formed by the N-terminal part of cl 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.

Author for correspondence: L. Paolozzi. Tel: + 39 06 72594325. Fax: + 39 06 2023500. e-mail: paolozzi@bio.uniroma2.it

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