Improved expression of kinases in Baculovirus-infected insect cells upon addition of specific kinase inhibitors to the culture helpful for structural studies.
As exemplified by three cases, we show that the addition of a small molecular weight inhibitor to the culture of Baculovirus-infected insect cells can dramatically improve the expression of a recombinant kinase. The expression of the tyrosine kinase KDR was sevenfold higher and mainly in a soluble form, when the KDR inhibitor PTK/ZK was added to the culture at the time of Baculovirus infection. The expression of the catalytic domain of the serine/threonine kinase PKCtheta, which is otherwise not possible with the Baculovirus expression system, was expressed mainly soluble at 120mg/L by the addition of the PKC inhibitor BIM XI to the culture of Baculovirus-infected insect cells. For Abl kinase, the expression could also be significantly increased by the addition of the Abl kinase inhibitor STI571 to the culture. For all three kinases, this method had previously been applied by us for the improved production of kinase/inhibitor complex protein, leading to the co-crystal structures. It is shown here at the cases KDR-PTK/ZK and PKCtheta-BIM XI, that the stimulatory effect of an inhibitor on kinase expression is applicable under many culture conditions. The presented method represents a valuable tool to obtain structural knowledge on kinase-inhibitor complexes.
Strauss
André
A
Novartis Pharma AG, NIBR, Discovery Technologies, CH-4002 Basel, Switzerland. andre.strauss@novartis.com
Fendrich
Gabriele
G
Horisberger
Michel André
MA
Liebetanz
Janis
J
Meyhack
Bernd
B
Schlaeppi
Jean-Marc
JM
Schmitz
Rita
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D008954:Models, Biological; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D011494:Protein Kinases; D016315:Proto-Oncogene Proteins c-abl