title: Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation.
abstract: Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.
authors.affiliation: Department of Haematological and Molecular Medicine, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute, King's College London, 123 Coldharbour Lane, London SE5 9NU, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Nesbeth
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Williams
authors.forename: Lucas
authors.initials: L
authors.lastname: Chan
authors.forename: Tony
authors.initials: T
authors.lastname: Brain
authors.forename: Nigel K H
authors.initials: NK
authors.lastname: Slater
authors.forename: Farzin
authors.initials: F
authors.lastname: Farzaneh
authors.forename: David
authors.initials: D
authors.lastname: Darling
mesh_terms: D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D008280:Magnetics; D008863:Microspheres; D008954:Models, Biological; D008979:Moloney murine leukemia virus / Q000235:genetics; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020800:Receptor, Nerve Growth Factor; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli