A cDNA clone for the catalytic subunit of murine cAMP-dependent protein kinase was placed into two expression vectors, pLWS-3 and pLSW-4. For pLWS-3, the entire coding region of the catalytic subunit was inserted into the NdeI site of pT7-7 under the control of the T7 promoter. pLWS-4 contains a polycistronic transcript under control of the lac UV5 promoter encoding for the type I regulatory subunit followed by the catalytic subunit. Significant expression was achieved with pLWS-4 in Escherichia coli 222 and JM101; however, the catalytic subunit was produced in an insoluble form. In the case of the catalytic subunit produced in E. coli BL21(DE3) by pLWS-3, the catalytic subunit accounted for approximately 30% of the total bacterial protein. Up to 5 mg of this catalytic subunit per liter of culture was in the soluble extract. Solubility was improved substantially when induction was carried out at 30 degrees C instead of 37 degrees C. This recombinant catalytic subunit was purified by phosphocellulose chromatography, followed by ammonium sulfate precipitation and gel filtration. A Mr of 38,000 was estimated based on size exclusion chromatography and on polyacrylamide gel electrophoresis. The recombinant protein had a free alpha-amino-terminal Gly in contrast to the mammalian enzyme which is myristylated at the amino-terminal glycine. The lack of acylation did not significantly alter the activity of the enzyme. The specific activity of 19 mumol/min/mg is comparable to the mammalian enzyme. The Km values for Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (43 microM) and MgATP (18.5 microM also were comparable. The absence of the acyl group also did not prevent holoenzyme formation. Holoenzyme activation by cAMP was indistinguishable for holoenzyme made with mammalian catalytic subunit and recombinant catalytic subunit. The recombinant enzyme was more sensitive than the mammalian enzyme to heat denaturation at 49 degrees C. The t1/2 for the recombinant catalytic subunit was 0.7 min in contrast to 3.9 min for the mammalian enzyme. This difference in stability may be attributable to the lack of the acyl group. The recombinant enzyme was particularly sensitive to heat denaturation in the presence of low concentrations (0.01%) of Triton X-100.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|