High yield purification of functional baculovirus vectors by size exclusion chromatography.
Recombinant baculoviruses carrying mammalian expression cassettes or "BacMam" are promising gene delivery vehicles shown to transduce mammalian cells efficiently both in vitro and in vivo. These viruses are vectors of choice because they are non-pathogenic; able to accommodate large foreign DNA inserts and can be produced at high titers. Hence, the demand for pure and functional baculovirus vectors for gene delivery experiments is anticipated in the future. The main goal of this work is to develop a simple and efficient process to purify recombinant baculovirus derived from Autographa californica multiple nucleopolyhedrovirus from a culture supernatant by size exclusion chromatography. The final yields obtained for total and infectious particles were 1.39 x 10(11) and 1.02 x 10(10) and recoveries of 25% and 24%, respectively. The virus was purified from the majority of the protein contaminants as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negative stain electron microscopy demonstrated that >95% of the purified virus was intact particles with shape like rod and average diameter and length of 60 and 266 nm, respectively. Transduction of 293 human embryonic kidney cells by a purified GFP-expressing BacMam at a multiplicity of transduction of 200 resulted in 36% positive cell population.
Transfiguracion
Julia
J
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Jorio
Hasnaa
H
Meghrous
Jamal
J
Jacob
Danielle
D
Kamen
Amine
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D002460:Cell Line; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D005374:Filtration / Q000379:methods; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D008851:Micropore Filters; D009036:Moths / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D011995:Recombination, Genetic; D018411:Spodoptera; D014161:Transduction, Genetic