title: Affinity analysis and application of dipeptides derived from l-tyrosine in plasmid purification.
abstract: The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions.
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: Soraia
authors.initials: S
authors.lastname: Ferreira
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: CICS-UBI, Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: Joana F A
authors.initials: JFA
authors.lastname: Valente
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, 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: Fani
authors.initials: F
authors.lastname: Sousa
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: João 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: Carla
authors.initials: C
authors.lastname: Cruz
mesh_terms: D002853:Chromatography, Liquid; D004151:Dipeptides / Q000032:analysis* / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D010957:Plasmids / Q000302:isolation & purification*; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000032:analysis* / Q000737:chemistry*
chemical_list: D004151:Dipeptides; D014443:Tyrosine
keywords: Chromatography; Nuclear magnetic resonance; Plasmid DNA; Surface plasmon resonance; l-tyrosine dipeptides