PMC:4103454 / 22810-23620 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4103454","sourcedb":"PMC","sourceid":"4103454","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4103454","text":"Protein Preparation\nClones of psNUC, stNUC, and saNUC (Uniprot accession numbers: A5WF35, A7J283, and A3EZR7, respectively) were synthesized by GeneART and subcloned into pGEX 6P-1. Human, mouse, and Pseudomonas aeruginosa FAN1 clones in pGEX 6P-1 were prepared by the DNA-cloning service at the University of Dundee. Proteins were expressed as glutathione S-transferase fusions in E. coli and purified by means of glutathione-Sepharose affinity chromatography. Proteins were cleaved off the affinity column with 3C protease and applied to Superdex 75 or 200 gel filtration in 300 mM NaCl, 20 mM Tris (pH 8), and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), depending on the target protein size. FAN1 protein purification required an additional heparin step to remove DNA contamination prior to gel filtration.","divisions":[{"label":"Title","span":{"begin":0,"end":19}}],"tracks":[]}