Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells.
Rapid expression of recombinant proteins for structure determination is one of the major challenges in pharmaceutical and academic research, since the number of potential drug targets has increased significantly in the last decade. Despite the fact that the baculovirus expression vector system is widely used for this purpose, the system is hampered by three very slow and tedious procedures, namely generation of high titer baculovirus stock, determination of the virus titer and discovery of the best conditions for protein expression. We herein describe the development of the ultraBac system to address and overcome these issues for protein expression in insect cells. We have established a new baculovirus expression technology for insect cells that is based on co-expression of GFP with target genes, a new regime for cell culturing and a highly efficient purification and enrichment procedure for recombinant baculovirus particles. Co-expression of GFP is used to monitor the infection of insect cells, to simplify titer determination and to optimize expression conditions. The new regime for cell culturing with increased viability of non-infected insect cells and its combination with the massive enrichment of virus particles via high-speed centrifugation enables the production of large amounts of recombinant virus in a very short period of time. By combining these techniques and by using the bicistronic vector pUltraBac-1, we have been able to cut the time-lines for protein expression in insect cells by half, approaching those for protein production in Escherichia coli. This new expression system is a significant step forward towards industrialized protein production in both, industry and academia.
Philipps
Björn
B
Discovery Technologies, Novartis Institutes for BioMedical Research, Basel, Switzerland.
Rotmann
Daniel
D
Wicki
Micha
M
Mayr
Lorenz M
LM
Forstner
Michael
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D018411:Spodoptera; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics; D000099157:Sphingosine Kinase
D049452:Green Fluorescent Proteins; D053592:Interleukin-1 Receptor-Associated Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000099157:Sphingosine Kinase; C485184:enhanced green fluorescent protein