RAP30 and RAP74 are subunits of RAP30/74 (TFIIF, beta gamma), a general initiation and elongation factor for transcription by RNA polymerase II. Methods were previously published for production of human RAP30 and RAP74 in bacterial cells, using a bacteriophage T7 promoter expression system. The vectors described for production of RAP74 were not very efficient and produced significant quantities of RAP74 amino terminal fragments. To improve these vectors, a segment of the human RAP74 cDNA was recoded using a preferred set of codons for translation in Escherichia coli. Recoding dramatically improved protein production and suppressed production of amino-terminal fragments. Improved vectors are reported that produce RAP74 with an LEHHHHHH carboxy-terminal extension (RAP74-H6), for purification on a Ni(2+)-affinity column, and also with the native carboxy terminus (RAP74). Methods for purification of RAP74-H6 and RAP74 are reported. Using these improved vectors, approximately 30 mg of soluble and active RAP74-H6 or RAP74 can be produced and purified from 1 liter of E. coli culture, representing a 10-fold improvement in protein production. Methods have also been developed for reconstitution of native RAP30/74 complex using recombinant proteins. This complex has indistinguishable activity from human RAP30/74 for accurate transcription in vitro.
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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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