Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α

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.

Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity

“…The hydrolysis of gliadins under the action of cysteine peptidases from sprouted wheat seeds Triticum aestivum triticain-α (Triticain-α) was also studied [34,100,101]. A form of the truncated enzyme Triticain-α, lacking a signal sequence and a granulin domain (Triticainα-GM), was recombinantly expressed in the bacterial system of E. coli, and hydrolyzed gliadins in a wide pH range from 3.0 to 6.5.…”

Peptidyl Fluoromethyl Ketones and Their Applications in Medicinal Chemistry

“…The latest outcomes on m-PFMKs as Cats inhibitors come from the thorough study of Rudzińska M. et al [ 21 ]. The authors developed two tetrapeptidyl m-FMKs, i.e., Ac-Pro-Leu-Val-Glu(OMe)-CH 2 F (Ac-PLVE-FMK) and Ac-Val-Leu-Pro-Glu(OMe)-CH 2 F (Ac-VLPE-FMK), on the basis of the well-known substrate Ac-PLVQ of the triticain-α, a wheat-derived ( Triticum aestivum ) cysteine protease of the papain-like family [ 22 ]. Both m-PFMKs were able to efficiently inhibit the activity of Cat-B and Cat-L in the target-based assay (human recombinant enzymes) and, more importantly, in the cell-based assay on two human renal cancer cell lines (769-P and A498), implying their ability to penetrate the cell membrane.…”

Redox-Mediated Post-Translational Modifications of Proteolytic Enzymes and Their Role in Protease Functioning

“…PLCPs are also susceptible to oxidation by H 2 O 2 . Triticain-α is a PLCP from Triticum aestivum L that has glutenase and collagenase activity and is believed to participate in seed maturation by digesting storage proteins during germination [44,45]. It was recently shown in our laboratory that triticain-α is inhibited by H 2 O 2 [46].…”