Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization
Monika Cserjan‐Puschmann, Nico Lingg, Petra Engele, Christina Kröß, Julian Loibl, Andreas Fischer, Florian Bacher, Anna‐Carina Frank, Christoph Öhlknecht, Cécile Brocard, Chris Oostenbrink, Matthias Berkemeyer, Rainer Schneider, Gerald Striedner, Alois Jungbauer
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.