Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling.
Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.
Chen
Rongjun
R
Centre for Molecular Nanoscience, School of Chemistry, University of Leeds, Leeds LS2 9JT, UK.
Folarin
Najeem
N
Ho
Vincent H B
VH
McNally
David
D
Darling
David
D
Farzaneh
Farzin
F
Slater
Nigel K H
NK
D000599:Amino Acids, Diamino / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry
D000599:Amino Acids, Diamino; C004720:7,8-diaminopelargonic acid; D001710:Biotin; C004749:desthiobiotin; D019809:Streptavidin