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

Triticain-α is a papain-like cysteine protease from wheat ( 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 sele...

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Bibliographic Details
Published in:	International journal of molecular sciences Vol. 18; no. 7; p. 1395
Main Authors:	Gorokhovets, Neonila V, Makarov, Vladimir A, Petushkova, Anastasiia I, Prokopets, Olga S, Rubtsov, Mikhail A, Savvateeva, Lyudmila V, Zernii, Evgeni Yu, Zamyatnin, Jr, Andrey A
Format:	Journal Article
Language:	English
Published:	Switzerland MDPI 29-06-2017
Subjects:	Bacteria - growth & development Bacteria - metabolism Catalytic Domain - genetics Cysteine Proteases - genetics Cysteine Proteases - metabolism DNA, Plant - genetics Escherichia coli - genetics Escherichia coli - metabolism Gene Expression Regulation Genes, Plant - genetics Histidine - metabolism Inclusion Bodies - metabolism Papain - genetics Papain - metabolism Peptide Hydrolases - genetics Peptide Hydrolases - metabolism Pichia - genetics Plant Proteins - genetics Plant Proteins - metabolism Protein Folding Proteolysis Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Solubility Temperature Triticum - enzymology Triticum - genetics celiac disease protein folding proteolytic cleavage autocatalytic activation recombinant protein papain-like cysteine protease
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Summary:	Triticain-α is a papain-like cysteine protease from wheat ( 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 ( ) strain GS115 and bacterial ( ) 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1422-0067 1422-0067
DOI:	10.3390/ijms18071395