Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display

Cláudia S. M. Fernandes, Inês Barbosa, Rute Castro, Ana Sofia Fidalgo Pombo Mendes Pina, Ana Sofia Coroadinha, Ana Barbas, A Cecília A Roque

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Retroviral particles are expensive to manufacture, mostly due to the downstream processing steps which result in low recoveries (≈30%) and concentration factors. In this work, a dodecapeptide phage-display library was panned against retrovirus like particles expressing the envelope protein Ampho4070A (VLPs-AMPHO) and VLPs without the target protein, used as a negative control (VLPs). A depletion/selection panning protocol was successfully used to deal with the structural complexity of the target, and a total of three distinct peptide sequences displaying preferential binding towards VLPs-AMPHO were found. Peptide 3 (CAAALAKPHTENHLLT), which appeared as one lead candidate, was synthesized and immobilized onto two purification matrices, cross-linked agarose and magnetic particles. The matrices selectively bound VLPs-AMPHO and in both cases recovery yields higher than 90% were obtained when employing mild elution conditions, while maintaining viral particle morphology and size.

Original languageEnglish
Pages (from-to)1513-1524
Number of pages12
JournalBiotechnology Journal
Volume11
Issue number12
DOIs
Publication statusPublished - 1 Dec 2016

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Bacteriophages
Peptides
Retroviridae
Particle Size
Virion
Sepharose
Libraries
Proteins

Keywords

  • Affinity ligands
  • Peptides
  • Phage display
  • Virus-like particles
  • VLP purification

Cite this

@article{f6a770fa08854b43858c8cddfa3f5e3f,
title = "Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display",
abstract = "Retroviral particles are expensive to manufacture, mostly due to the downstream processing steps which result in low recoveries (≈30{\%}) and concentration factors. In this work, a dodecapeptide phage-display library was panned against retrovirus like particles expressing the envelope protein Ampho4070A (VLPs-AMPHO) and VLPs without the target protein, used as a negative control (VLPs). A depletion/selection panning protocol was successfully used to deal with the structural complexity of the target, and a total of three distinct peptide sequences displaying preferential binding towards VLPs-AMPHO were found. Peptide 3 (CAAALAKPHTENHLLT), which appeared as one lead candidate, was synthesized and immobilized onto two purification matrices, cross-linked agarose and magnetic particles. The matrices selectively bound VLPs-AMPHO and in both cases recovery yields higher than 90{\%} were obtained when employing mild elution conditions, while maintaining viral particle morphology and size.",
keywords = "Affinity ligands, Peptides, Phage display, Virus-like particles, VLP purification",
author = "Fernandes, {Cl{\'a}udia S. M.} and In{\^e}s Barbosa and Rute Castro and Pina, {Ana Sofia Fidalgo Pombo Mendes} and Coroadinha, {Ana Sofia} and Ana Barbas and Roque, {A Cec{\'i}lia A}",
note = "Sem PDF conforme despacho. info:eu-repo/grantAgreement/FCT/5876/147258/PT# info:eu-repo/grantAgreement/FCT/3599-PPCDT/118317/PT# info:eu-repo/grantAgreement/FCT/SFRH/SFRH{\%}2FBPD{\%}2F72523{\%}2F2010/PT# This work was supported by the Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The authors thank Fundacao para a Ciencia e a Tecnologia, Portugal, for the project PTDC/EBB-BIO/118317/2010, and research fellowships PD/BD/105871/2014 and SFRH/BPD/72523/2010 for C. Fernandes and R. Castro, respectively The authors thank Hello Tomas (ITOB/IBET, Portugal) for providing pMONO-zeo-4070A plasmid. The authors acknowledge the Electron Microscopy Unit from Institute Gulbenkian de Ciencia for the TEM analysis.",
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Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display. / Fernandes, Cláudia S. M.; Barbosa, Inês; Castro, Rute; Pina, Ana Sofia Fidalgo Pombo Mendes; Coroadinha, Ana Sofia; Barbas, Ana; Roque, A Cecília A.

In: Biotechnology Journal, Vol. 11, No. 12, 01.12.2016, p. 1513-1524.

Research output: Contribution to journalArticle

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AU - Fernandes, Cláudia S. M.

AU - Barbosa, Inês

AU - Castro, Rute

AU - Pina, Ana Sofia Fidalgo Pombo Mendes

AU - Coroadinha, Ana Sofia

AU - Barbas, Ana

AU - Roque, A Cecília A

N1 - Sem PDF conforme despacho. info:eu-repo/grantAgreement/FCT/5876/147258/PT# info:eu-repo/grantAgreement/FCT/3599-PPCDT/118317/PT# info:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBPD%2F72523%2F2010/PT# This work was supported by the Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). The authors thank Fundacao para a Ciencia e a Tecnologia, Portugal, for the project PTDC/EBB-BIO/118317/2010, and research fellowships PD/BD/105871/2014 and SFRH/BPD/72523/2010 for C. Fernandes and R. Castro, respectively The authors thank Hello Tomas (ITOB/IBET, Portugal) for providing pMONO-zeo-4070A plasmid. The authors acknowledge the Electron Microscopy Unit from Institute Gulbenkian de Ciencia for the TEM analysis.

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AB - Retroviral particles are expensive to manufacture, mostly due to the downstream processing steps which result in low recoveries (≈30%) and concentration factors. In this work, a dodecapeptide phage-display library was panned against retrovirus like particles expressing the envelope protein Ampho4070A (VLPs-AMPHO) and VLPs without the target protein, used as a negative control (VLPs). A depletion/selection panning protocol was successfully used to deal with the structural complexity of the target, and a total of three distinct peptide sequences displaying preferential binding towards VLPs-AMPHO were found. Peptide 3 (CAAALAKPHTENHLLT), which appeared as one lead candidate, was synthesized and immobilized onto two purification matrices, cross-linked agarose and magnetic particles. The matrices selectively bound VLPs-AMPHO and in both cases recovery yields higher than 90% were obtained when employing mild elution conditions, while maintaining viral particle morphology and size.

KW - Affinity ligands

KW - Peptides

KW - Phage display

KW - Virus-like particles

KW - VLP purification

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DO - 10.1002/biot.201600025

M3 - Article

VL - 11

SP - 1513

EP - 1524

JO - Biotechnology Journal

JF - Biotechnology Journal

SN - 1860-6768

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