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.

BACKGROUND: Recombinant proteins produced for use as biopharmaceuticals need to harbor their native N-terminus. A drawback in expression of recombinant proteins as fusion proteins with an affinity fusion-tag is that enzymatic or chemical processing is required to trim the artificial tag and release the true protein of interest. In many cases, however, this processing step generates an incorrect N-terminus.RESULTS: Human fibroblast growth factor 2 (FGF2) was expressed as a fusion protein in Escherichia coli fed-batch cultivations. The protein of interest (POI) carried an N-terminal affinity fusion-tag which enabled purification via affinity chromatography. After enzymatic removal of the affinity fusion-tag with a circularly permuted human caspase-2 (cpCasp2), the POI was further purified using subtractive affinity chromatography. Mass spectrometric analysis confirmed the authentic N-terminus of the POI. The generated POI was highly pure with 42 ppm host cell protein, 3.7 ∼g mL −1 dsDNA and ∼ 1000 EU mL −1 endotoxin. Only a small number of E. coli host cell proteins were co-purified with the POI. Because of the high specificity of the novel protease cpCasp2, no off-target cleavage could be observed. CONCLUSION: Our findings demonstrate that cpCasp2 can be used for the production of native proteins using a fusionprotein process. This represents a first case study at large laboratory scale for the production of an industrially relevant protein. This technology constitutes the basis of a highly scalable cpCasp2-based platform fusion protein process (CASPON technology) purification platform.

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.