title: L-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification.
abstract: The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three l-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of l-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (KD=10(-9) and 10(-8)M) and the conditions tested show that the use of HEPES 100mM enables the separation and purification of supercoiled at T=10°C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to l-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and π-π stacking. The chromatography experiments are performed with l-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10°C by decreasing the concentration of (NH4)2SO4 from 2.7 to 0M in HEPES for pVAX-LacZ and 2.65M to 0M in HEPES for pPH600. Overall, l-tryptophan derivatives can be a promising strategy to purify supercoiled for pharmaceutical applications.
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Tiago
authors.initials: T
authors.lastname: Santos
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Josué
authors.initials: J
authors.lastname: Carvalho
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; CENIMAT/I3N, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Marta C
authors.initials: MC
authors.lastname: Corvo
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Eurico J
authors.initials: EJ
authors.lastname: Cabrita
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.affiliation: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Electronic address: carlacruz@fcsaude.ubi.pt.
authors.forename: C
authors.initials: C
authors.lastname: Cruz
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D004151:Dipeptides / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D010957:Plasmids; D012685:Sepharose / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D004278:DNA, Superhelical; D004151:Dipeptides; D009711:Nucleotides; D014364:Tryptophan; D012685:Sepharose
keywords: Biorecognition; Supercoiled purification; l-tryptophan dipeptides