A NOVEL BACTERIAL VECTOR SYSTEM FOR MONITORING PROTEIN-PROTEIN INTERACTIONS IN THE CAMP-DEPENDENT PROTEIN-KINASE COMPLEX

A bacterial expression vector is described for investigation of protei n-protein interactions. Important features of the vector include parti tion of the cI repressor of bacteriophage lambda into two functional d omains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding dom ain of cI. Two different reporter systems have been employed; expressi on of either a suppressor tRNA or the alkaline phosphatase gene is dep endent in both cases on the extent of repression of the major leftward promoter of lambda (lambda P-L). The cAMP-dependent protein kinase (P KA) has been used as a model protein complex because both homodimer an d heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker re striction sites still functions as a repressor. Substitution of the di merisation domain of the cI repressor with the regulatory subunit of P KA does not diminish the ability of a cI fusion protein to repress exp ression of the reporter gene from lambda P-L, indicating that the regu latory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. C omplete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reco nstituted in the E. coli cytoplasm, where all subunit interactions app ear functional and respond as expected to the allosteric modulator cAM P.