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Int. J. Mol. Sci. 2017, 18(7), 1395; doi:10.3390/ijms18071395

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

1
Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Trubetskaya str., 8, bld. 2, Moscow 119991, Russia
2
Lomonosov Moscow State University, Faculty of Biology, Moscow 119991, Russia
3
International Associated Laboratory (LIA) 1066 French-Russian Joint Cancer Research Laboratory, Villejuif 94805, France
4
Institut für Pharmakologie and Klinische Pharmazie, Philipps-Universität Marburg, Marburg D-35043, Germany
5
Department of Biological Chemistry, Sechenov First Moscow State Medical University, Trubetskaya str., 8, bld. 2, Moscow 119991, Russia
6
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia
*
Author to whom correspondence should be addressed.
Received: 23 May 2017 / Revised: 21 June 2017 / Accepted: 23 June 2017 / Published: 29 June 2017
(This article belongs to the Special Issue Recombinant Proteins)
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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. View Full-Text
Keywords: recombinant protein; papain-like cysteine protease; protein folding; proteolytic cleavage; autocatalytic activation; celiac disease recombinant protein; papain-like cysteine protease; protein folding; proteolytic cleavage; autocatalytic activation; celiac disease
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Gorokhovets, N.V.; Makarov, V.A.; Petushkova, A.I.; Prokopets, O.S.; Rubtsov, M.A.; Savvateeva, L.V.; Zernii, E.Y.; Zamyatnin Jr., A.A. Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α. Int. J. Mol. Sci. 2017, 18, 1395.

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