Vectors for recombinational cloning and gene expression in mammalian cells using modified vaccinia virus Ankara.
Modified vaccinia virus Ankara (MVA) is a safe vector for high-level expression of proteins in mammalian cells. To simplify the molecular cloning procedures for shuttling genes into the MVA genome, we constructed generic destination plasmids that allow in vitro recombinational cloning (Gateway) and quick isolation of expression plasmids for any gene to be incorporated into the virus. Downstream purification steps were simplified by including N-terminal peptide tags (His, Strep, and Flag) in the generic plasmids. We demonstrate the ability to produce 10mg of beta-glucuronidase from 10(8) hamster cells and to purify tagged proteins with affinity gels.
Pradeau-Aubreton
Karine
K
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Structural Biology and Genomics, CNRS UMR, INSERM U, University of Strasbourg, Illkirch, France.
Ruff
Marc
M
Garnier
Jean-Marie
JM
Schultz
Patrick
P
Drillien
Robert
R
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D015870:Gene Expression; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D011994:Recombinant Proteins; D005966:Glucuronidase