title: Selective purification of supercoiled p53-encoding pDNA with L-methionine-agarose matrix.
abstract: The p53 tumor suppressor gene has been widely explored for gene therapy as an alternative to the common treatments. Recently, the supercoiled conformation of a p53-encoding plasmid proved to be more efficient in cell transfection and protein expression than the open circular conformation. To successfully isolate this isoform, several chromatographic techniques have been used, namely affinity chromatography with amino acids as ligands. However, the study of new matrices and ligands with higher specificity and robustness for supercoiled plasmid purification is still required. The present work explores for the first time a new matrix of l-methionine-agarose to efficiently purify the supercoiled p53-encoding plasmid. The binding/elution conditions, such as salt concentration and temperature, were manipulated and combined to attain the best strategy. Therefore, the supercoiled plasmid isoform was purified from a clarified lysate by using a decreasing stepwise gradient comprising 2.35 and 1.7M ammonium sulfate in 10mM Tris-HCl, pH 8.0, and finally 10mM Tris-HCl, pH 8.0, at 5°C. After accomplishing the purification process, we performed several tests to assess the quality of the supercoiled plasmid, revealing that the amounts of proteins, gDNA, RNA, and endotoxins were significantly reduced or undetectable in the final formulation.
authors.affiliation: Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal.
authors.forename: J F A
authors.initials: JF
authors.lastname: Valente
authors.affiliation: Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Sousa
authors.affiliation: Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal.
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.affiliation: Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal. Electronic address: fani.sousa@fcsaude.ubi.pt.
authors.forename: F
authors.initials: F
authors.lastname: Sousa
mesh_terms: D002845:Chromatography / Q000379:methods*; D004278:DNA, Superhelical / Q000235:genetics* / Q000302:isolation & purification*; D023281:Genomics; D008715:Methionine / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D004278:DNA, Superhelical; D016159:Tumor Suppressor Protein p53; D012313:RNA; D012685:Sepharose; D008715:Methionine
keywords: Affinity chromatography; Biorecognition; Supercoiled isoform; l-Methionine; p53-encoding plasmid