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.
Highlights
A perfect protease for fusion tag removal has not yet been found
The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity
We describe how to arrive close to such a perfect protease by circularly permuting human caspase-2
Summary
A perfect protease for fusion tag removal has not yet been found. Such enzymes should be highly specific, soluble, and stable and should be easy to manufacture to reduce the economic burden. Platform technologies could accelerate the development efforts and time and reduce costs for laboratory and industrial production of proteins [1] Such technologies should ideally combine efficient soluble expression, an affinity tag enabling fast and simple purification and a system for efficient tag removal. We describe (i) the generation of a circularly permuted caspase-2, (ii) the effect of a solubility tag on expression level, (iii) an optimized model-based controlled fermentation process, (iv) a swift and efficient purification process of the generated enzyme, and (v) the enzyme characteristics with respect to kinetics and stability. A constitutively active cpCasp could be an ideal protease for biotechnological applications Such a variant can be generated through circular permutation (cp), which is the covalent linkage of native N- and C-terminus and the introduction of new termini elsewhere in the protein [34]. Published examples of circularly permuted caspases focused on researching apoptosis and did not have a biotechnological use in mind, aiming for a structure similar to the wild type variants
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