title: A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport.
abstract: In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.
authors.affiliation: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Kijung
authors.initials: K
authors.lastname: Sung
authors.forename: Michael T
authors.initials: MT
authors.lastname: Maloney
authors.forename: Jingkun
authors.initials: J
authors.lastname: Yang
authors.forename: Chengbiao
authors.initials: C
authors.lastname: Wu
mesh_terms: D000818:Animals; D001369:Axons / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005727:Ganglia, Spinal / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology*; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D020932:Nerve Growth Factor; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli