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 10 mg of β-glucuronidase from 10⁸ hamster cells and to purify tagged proteins with affinity gels.