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 protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and 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 restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory 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. Complete 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 reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.
Cairns
M T
MT
Department of Medicine, University of Cambridge, UK. mike@jupiter.qub.ac.uk
Green
A J
AJ
White
P M
PM
Johnston
P G
PG
Brenner
S
S
D000469:Alkaline Phosphatase / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011494:Protein Kinases; D012343:RNA, Transfer / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D012343:RNA, Transfer; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase