The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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