Abstract
Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichia pastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.
Highlights
Recombinant proteases of exogenous origins are broadly used for therapeutic applications [1]
Keeping in mind the outstanding time and cost efficiency of protein production using bacterial expression systems, our primary efforts were aimed at optimizing domain structure of Triticain-α to increase solubility of the respective product in E. coli
HSP70 itself was produced in E. coli BL21 (DE3) cells as a cytoplasmic protein, it induced no transfer of Triticain-α-CatD into the soluble fraction (Table 2)
Summary
Recombinant proteases of exogenous origins are broadly used for therapeutic applications [1]. Papain-like proteases are no exception since most attempts to perform their heterologous expression in E. coli resulted in production of the recombinant protein in insoluble form [12] Their in vitro refolding is commonly associated with simultaneous maturation of an active enzyme, which undergoes autocatalytic degradation. The obtaining of the active enzyme mandatory included combination of Ni-NTA chromatography of the denaturated protein and its subsequent refolding, which, as noted above, is a time-consuming process associated with autocatalytic activation of the enzyme, and can be poorly controlled Considering all these observations, in the present study, we have focused on the rational design of the optimal domain architecture of Triticain-α and selection of expression system to develop potentially scalable protocol for cheap and efficient production of active recombinant enzyme
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