期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 56, 期 2, 页码 167-176出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.06.008
关键词
Ab1 kinase; baculovirus; crystallization; expression; KDR; kinase inhibitor; PKC theta
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 PKC theta, which is otherwise not possible with the Baculovirus expression system, was expressed mainly soluble at 120 mg/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 ST1571 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 PKC theta-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. (C) 2007 Elsevier Inc. All rights reserved.
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