TY - JOUR
T1 - Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display
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.
PY - 2016/12/1
Y1 - 2016/12/1
N2 - 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.
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
U2 - 10.1002/biot.201600025
DO - 10.1002/biot.201600025
M3 - Article
C2 - 27491899
SN - 1860-6768
VL - 11
SP - 1513
EP - 1524
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 12
ER -