Engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells.
One of the most common methods for producing recombinant baculovirus for insect cell protein production involves a transposition mediated system invented over 2 decades ago. This Tn7-mediated system, commercially sold as Bac-to-Bac, has proven highly useful for construction of high quality baculovirus, but suffers from a number of drawbacks which reduce the efficiency of the process and limit its utility for high throughput protein production processes. We describe here the creation of Bac-2-the-Future, a 2nd generation Tn7-based system for recombinant baculovirus production which uses optimized expression vectors, new E. coli strains, and enhanced protocols to dramatically enhance the efficiency of the baculovirus production process. The new system which we describe eliminates the need for additional screening of positive clones, improves the efficiency of transposition, and reduces the cost and time required for high throughput baculovirus production. The system is compatible with multiple cloning methodologies, and has been demonstrated to produce baculovirus with equal or better titer and protein productivity than the currently available systems.
Protein Expression Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702, United States.
Jennifer L
JL
Mehalko
Protein Expression Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc. PO Box B, Frederick, MD 21702, United States. Electronic address: dom.esposito@nih.gov.
Dominic
D
Esposito
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D014162:Transfection
D011994:Recombinant Proteins
Bac-to-Bac; Baculovirus; Insect cells; Protein production; Tn7 transposition