title: Malonate-assisted purification of human caspases.
abstract: Caspases have in the past decade become some of the most intensely pursued targets for the design of small-molecule inhibitors. Two significant technological roadblocks to developing caspase-binding molecules are the poor solubility of a subset of the bacterially expressed proteins and the instability of the renatured proteins that results from rapid inactivating autolysis at high protein concentrations. In this report, we present a generalized method of renaturing human caspases and inhibiting the self-proteolytic activity of the enzymes without a need for covalent active-site inhibitors. Our method, which involves blocking the S1 region of the active site with malonate, enables one to inhibit fully the inactivating autolysis in human caspases and increases the yields of renatured active enzyme. It furthermore does not necessitate removal of malonate prior to setting up enzymatic assays since as high as 100-mM concentrations of malonate do not compete efficiently with caspase substrates or larger caspase inhibitors for binding to the active site. The method described in this report simplifies greatly caspase purification and makes it possible to stabilize the enzymes against autolysis without a need for costly, and frequently synthetically challenging, small-molecule inhibitors.
authors.affiliation: Department of Biology, Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Scheer
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Romanowski
mesh_terms: D001329:Autolysis; D020170:Caspase 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020134:Catalytic Domain; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008314:Malonates / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008314:Malonates; D021122:Protein Subunits; D011994:Recombinant Proteins; C030290:malonic acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1