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US 5,474,921 AGranted
Expression and purification of phosphoinositide-specific phospholipase C-.gamma.
AssigneeMerck & Co., Inc.
InventorsKenneth S. Koblan, David L. Pompliano
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Abstract
The present invention is directed to an assay which is used to determine the inhibitory activity of a test compound against a particular phosphoinositide-specific phospholipase C enzyme, that enzyme being phospholipase C.gamma.. The present invention is also directed to the preparation of phospholipase C.gamma. by recombinant expression in a bacterial cell and isolation of the expressed enzyme.
Claims1
A method of expressing and isolating mammalian phospholipase C.gamma..sub.1 in an enzymatically active form which comprises the steps of:
- a) transforming bacterial host cells with a plasmid that comprises a cDNA which encodes said mammalian phospholipase C.gamma..sub.1, wherein the cDNA has the nucleotide sequence of SEQ.ID.NO.:
- 1, and a second eDNA operationally linked to the eDNA of SEQ.ID.NO.:
- 1, said second eDNA which encodes for the epitope tag GluGluPhe, said epitope tag which is incorporated at the C-terminus of the phospholipase C.gamma..sub.1 when it is expressed;
- b) growing and harvesting the transformed host cells;
- c) exposing the harvested host cells to an affinity column containing an antibody which specifically binds said epitope tag;
- and
- d) eluting said affinity column to provide isolated enzymatically active mammalian phospholipase C.gamma..sub.1.
4 more elements
Dependencies are inferred from the claim text.