Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.
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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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