Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies

Abstract

The interaction of two Escherichia coli transcription factors (sigma 54 and sigma 32) with the core RNA polymerase was studied here. We examined the core binding ability of various fragments of these two transcription factors using a novel method for the in vitro synthesis of truncated proteins in a coupled transcription-translation (S-30) system. The method uses DNA templates generated by the polymerase chain reaction to direct synthesis of precisely truncated fragments of the encoded protein. Primers for the polymerase chain reaction contain transcription and translation signals so that the resulting product can be incubated in the S-30 directly. The synthesized proteins were used for mapping of both functional domains and epitopes of monoclonal antibodies to sigma 54 and sigma 32.