Development of a novel prokaryotic two-hybrid system for the detection and analysis of protein-protein interactions in vivo

摘要

The purpose of this work was to develop a novel prokaryotic two-hybrid (P2H) system for the detection of protein-protein interactions in vivo. The system is based on the transcriptional activation and DNA binding domains of the Sinorhizobium meliloti 2011 enhancer binding protein NifA. This protein is responsible for the transcriptional activation of several nif and fix genes, including the nifHDK operon, in response to cellular oxygen and/or nitrogen status.ududNifA is modular in nature and has three independently folded domains. The central domain of about 220 amino acids is responsible for the activation of transcription. The function of the Nterminal domain is unknown while the C-terminal domain contains a helix-turn-helix motif that is required for binding to a specific upstream activator sequence. PCR was used to amplify the individual domains of the NifA protein and also to amplify the nifH promoter. The PCR products obtained were used to construct suitable plasmids and reporter gene constructs for the P2H system.ududThree pKK223-3 based Prey plasmids were constructed using the N-terminal-central (NC) transcriptional activation domain of NifA. Cloning of a known gene or a library of DNA fragments into the multiple cloning site (MCS) of any of these plasmids generates hybrid proteins bearing the N terminal-central (NC) activation domain of NifA. The three Prey plasmids differed from each other only in the reading frame of the MCS relative to the upstream NifA activation domain sequence. The purpose of constructing these three plasmids was to facilitate library construction. Another pKK223-3 based Control plasmid expressing the whole NifA protein was also constructed.ududTwo pACYC184 based Bait plasmids were also constructed encoding the sequence for the Cterminal DNA binding domain of NifA. Cloning into the MCS of the Bait construct on either of the plasmids results in the expression of a hybrid protein bearing the DNA binding domain of NifA. In addition to the Bait construct these plasmids also maintained the reporter gene constructs. One of the Bait plasmids contained a nifH. lacZa reporter gene construct while the other contained a nifH:genf (gentamycin resistance) reporter gene construct. These Bait plasmids were compatible with the Prey plasmids and the Control plasmid.ududIn addition to the construction of the above mentioned plasmids suitable host strains of E. coli were developed in which the plasmids could be used. These strains had deletions of the glnG gene since the product of this gene, NtrC, had been observed to weakly activate the nifH promoter and could therefore contribute to false positives. Two glnG mutant strains of E. coli called ET6016 and YMC11 were obtained. These lacked the correct lacZa complementation background required for the nifH.lacZareporter system to function and so the correct genetic background was introduced into the two strains on an F ’ factor by conjugation to form strains ET6016r//F' and YMC1 ifF'.ududHaving constructed the appropriate plasmids, developed suitable strains and optimised growth conditions under which the novel NifA based P2H system would be used, the system was evaluated with the model protein-protein interaction between the appropriate domains of the intimin protein and the Tir receptor of enteropathagenic E. coli which had been demonstrated in a yeast two-hybrid system by another laboratory. The sequence for the Int280oc protein was cloned into the pPC229-A Prey plasmid to form plasmid pPC233 which expressed a hybrid protein consisting of the Int280a protein fused to the NifA transcription activating NC domain. The sequence for the Tir protein was cloned into the pPC187-A Bait plasmid to form plasmid pPC190 which expressed a hybrid protein consisting of the Tir protein fused to the Cterminal DNA binding domain of NifA. The plasmids pPC233 and pPC190 were cotransformed into the E. coli strain ET6016r/yF’ which was then plated on appropriate selective media to test for activation of the nifH:lacZa reporter gene. No interaction between Tir and Int280oc hybrid proteins was detected and the possible reasons for the failure to detect an interaction are discussed.