Targeting and purification of metabolically biotinylated baculovirus.
Targeting viral entry is one of the major goals in the development of vectors for gene therapy. Ideally, the coupling of each new targeting motif would not require changes in vector structure. To achieve this, we developed novel metabolically biotinylated baculoviral vectors by displaying a small biotin acceptor peptide (BAP) fused either to different sites in the baculovirus glycoprotein gp64 or to the transmembrane anchor of vesicular stomatitis virus G protein. Baculoviral particles were biotinylated during vector production by coexpression of Escherichia coli biotin ligase (BirA). The insertion of BAP at amino acid position 283 of gp64 resulted in the most efficient biotin display. Unlike vectors with lower biotin display, these vectors also showed improved transduction when retargeted to transferrin, epidermal growth factor, and CD46 receptors overexpressed on rat glioma and human ovarian carcinoma cells. Biotinylated baculoviral vectors could also be concentrated by one-step magnetic particle-based capture to reach titers up to 10(10) plaque-forming units/ml. These results demonstrate the utility of metabolically biotinylated baculovirus for vector targeting and viral purification applications.
Kaikkonen
Minna U
MU
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, FIN-70211 Kuopio, Finland.
Viholainen
Jenni I
JI
Närvänen
Ale
A
Ylä-Herttuala
Seppo
S
Airenne
Kari J
KJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000235:genetics; D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008280:Magnetics; D051916:Membrane Cofactor Protein / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry; D014161:Transduction, Genetic / Q000379:methods*; D014168:Transferrin / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053586:Virus Internalization
D029968:Escherichia coli Proteins; C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014168:Transferrin; D014759:Viral Envelope Proteins; D004815:Epidermal Growth Factor; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli