ABSTRACT
Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.
Subject(s)
Caspase 2/biosynthesis , Cysteine Endopeptidases/biosynthesis , Escherichia coli/chemistry , Recombinant Fusion Proteins/biosynthesis , Caspase 2/isolation & purification , Caspase 2/metabolism , Cloning, Molecular , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Escherichia coli/metabolism , Humans , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
Initiation of apoptosis by chemotherapeutic drugs is one of the most effective approaches to the treatment of cancers. Caspases, the main enzymes of apoptosis, undergo activation to initiate cell death. Activation of initiator caspases requires their binding to special protein complexes. For elucidation of the mechanisms of apoptosis, these complexes should be isolated. However, their purification is challenging because they are formed in the cell in negligible amounts and rapidly degrade. We have developed an effective way to isolate caspase activation complexes formed in tumor cells in response to DNA damage. The method is based on combination of gel filtration with immunoprecipitation. The first stage is aimed at the separation of the high-molecular-weight caspase activation complexes and their monomeric forms, which allows increasing the efficiency of isolation of complexes at the second stage.
Subject(s)
Caspases, Initiator/isolation & purification , DNA Damage/physiology , Apoptosis/genetics , Apoptosis/physiology , Blotting, Western , Caspase 2/isolation & purification , Caspase 2/metabolism , Caspase 8/isolation & purification , Caspase 8/metabolism , Caspases, Initiator/metabolism , Cell Line, Tumor , Chromatography, Gel , Cisplatin/pharmacology , Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , DNA Damage/drug effects , DNA Damage/genetics , Flow Cytometry , Humans , ImmunoprecipitationABSTRACT
Caspase-2 has been shown to function in apoptosis and in some non-apoptotic pathways, including tumor suppression and aging. Caspase-2 has some unique features and is the only caspase that constitutively localizes to the nucleus, although its nuclear function remains unknown. During apoptosis signaling, caspase-2 rapidly homodimerizes, which leads to its activation and proteolytic processing. The activation of caspase-2 can be measured by assessing its dimerization and/or cleavage of the caspase-2 zymogen and its substrates. This chapter outlines commonly used methods to purify recombinant caspase-2 and assess its activity and function in vitro and in cultured cells or tissue extracts.
