Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020186 | Protein Expression and Purification | 2016 | 8 Pages |
Abstract
Several protease IV enzymes are widely used in proteomic research. Specifically, protease IV from Pseudomonas aeruginosa has lysyl endopeptidase activity. Here, we report the recombinant expression, refolding, activation, and purification of this protease in Escherichia coli. Proteolytic instability of the activated intermediate, a major obstacle for efficient production, is controlled through ammonium sulfate precipitation. The purified protease IV exhibits superior lysyl endopeptidase activity compared to a commercial product.
Keywords
FCMPSMSTrxFASPTCLUPLCLC-MSTLCKDTTIPTGORFFDRLB mediumProtease IVSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisdissolved oxygenisopropyl thio-β-d-galactosideRefoldingRecombinant expressionfilter-aided sample preparationthioredoxindithiothreitolLuria-Bertani mediumcircular dichroismPseudomonas aeruginosaLiquid chromatography-mass spectrometrypeptide spectrum matchesactivityopen reading frameInclusion bodiesfalse discovery rateMolecular weightPurificationultra performance liquid chromatographyhigh performance liquid chromatographyHPLC
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Authors
Mingzhi Zhao, Man Cai, Feilin Wu, Yao Zhang, Zhi Xiong, Ping Xu,