Affinity analysis and application of dipeptides derived from L-tyrosine in plasmid purification

Soraia Ferreira, Josue Carvalho, Joana F. A. Valente, Marta C. Corvo, Eurico J. Cabrita, Fani Sousa, Joao A. Queiroz, Carla Cruz

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 (In) 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. (C) 2015 Elsevier B.V. All rights reserved.

Original languageEnglish
Pages (from-to)47-58
Number of pages12
JournalJournal of Chromatography B
Volume1006
DOIs
Publication statusPublished - 1 Dec 2015

Keywords

  • L-tyrosine dipeptides
  • Plasmid DNA
  • Surface plasmon resonance
  • Nuclear magnetic resonance
  • Chromatography
  • ANGLE-SPINNING NMR
  • HIGH-RESOLUTION
  • DNA PURIFICATION
  • SOLID SUPPORTS
  • CHROMATOGRAPHY
  • RECOGNITION
  • HISTIDINE
  • ARGININE
  • LIGAND
  • TRANSFECTION

Cite this

Ferreira, Soraia ; Carvalho, Josue ; Valente, Joana F. A. ; Corvo, Marta C. ; Cabrita, Eurico J. ; Sousa, Fani ; Queiroz, Joao A. ; Cruz, Carla. / Affinity analysis and application of dipeptides derived from L-tyrosine in plasmid purification. In: Journal of Chromatography B. 2015 ; Vol. 1006. pp. 47-58.
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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 (In) 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. (C) 2015 Elsevier B.V. All rights reserved.",
keywords = "L-tyrosine dipeptides, Plasmid DNA, Surface plasmon resonance, Nuclear magnetic resonance, Chromatography, ANGLE-SPINNING NMR, HIGH-RESOLUTION, DNA PURIFICATION, SOLID SUPPORTS, CHROMATOGRAPHY, RECOGNITION, HISTIDINE, ARGININE, LIGAND, TRANSFECTION",
author = "Soraia Ferreira and Josue Carvalho and Valente, {Joana F. A.} and Corvo, {Marta C.} and Cabrita, {Eurico J.} and Fani Sousa and Queiroz, {Joao A.} and Carla Cruz",
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Affinity analysis and application of dipeptides derived from L-tyrosine in plasmid purification. / Ferreira, Soraia; Carvalho, Josue; Valente, Joana F. A.; Corvo, Marta C.; Cabrita, Eurico J.; Sousa, Fani; Queiroz, Joao A.; Cruz, Carla.

In: Journal of Chromatography B, Vol. 1006, 01.12.2015, p. 47-58.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Affinity analysis and application of dipeptides derived from L-tyrosine in plasmid purification

AU - Ferreira, Soraia

AU - Carvalho, Josue

AU - Valente, Joana F. A.

AU - Corvo, Marta C.

AU - Cabrita, Eurico J.

AU - Sousa, Fani

AU - Queiroz, Joao A.

AU - Cruz, Carla

N1 - Sem PDF. Carla Cruz acknowledges post-doctoral grant from FCT SFRH/BPD/100015/2014 and J.F.A. Valente also acknowledges a Ph.D. grant from FCT (Ref SFRH/BD/96809/2013). This work was supported by FCOMP-01-0124-FEDER-041068-EXPL/QEQ-MED/1068/2013.

PY - 2015/12/1

Y1 - 2015/12/1

N2 - 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 (In) 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. (C) 2015 Elsevier B.V. All rights reserved.

AB - 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 (In) 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. (C) 2015 Elsevier B.V. All rights reserved.

KW - L-tyrosine dipeptides

KW - Plasmid DNA

KW - Surface plasmon resonance

KW - Nuclear magnetic resonance

KW - Chromatography

KW - ANGLE-SPINNING NMR

KW - HIGH-RESOLUTION

KW - DNA PURIFICATION

KW - SOLID SUPPORTS

KW - CHROMATOGRAPHY

KW - RECOGNITION

KW - HISTIDINE

KW - ARGININE

KW - LIGAND

KW - TRANSFECTION

U2 - 10.1016/j.jchromb.2015.10.025

DO - 10.1016/j.jchromb.2015.10.025

M3 - Article

VL - 1006

SP - 47

EP - 58

JO - Journal of Chromatography B

JF - Journal of Chromatography B

SN - 1570-0232

ER -