Abstract
The proteolytic enzyme enteropeptidase (EC 3.4.21.9) is a glycoprotein consisting of a heavy and a light polypeptide chain. The light chain of the enzyme is serine protease whose recombinant variants are used in biotechnology for the cleavage of peptide bonds in hybrid proteins downstream of the specific recognition site DDDDK- or DDDDR-. We have developed a pilot method for producing the recombinant human enteropeptidase light chain containing a cascade of two affine tags. The expression plasmid vector рL-HEP-H6-CBD encoding the hybrid protein was constructed, which contains the sequence of the human enteropeptidase light chain and the metal- and chitin-binding domains. The vector was transformed into the Escherichia coli BL21(DE3) strain, and, after the expression of the hybrid protein HEP-H6-CBD, a relatively simple scheme of renaturation and purification of the enzyme was developed. After the immobilization of the purified preparation on a chitin carrier, the enzyme retained the activity of specific cleavage of substrates for at least six cycles.
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Abbreviations: mHP-1, model hybrid protein 1 (Trx-interferon alpha-2b); mHP-2, model hybrid protein 2 (Trx-purotoxin-6); L-HEP, the light chain of human enteropeptidase; t-L-HEP, the human enteropeptidase light chain L-HEP–H6–CBD containing a cascade of two affine tags; i-t-L-HEP, an immobilized variant of the human enteropeptidase light chain L-HEP–H6–CBD containing a cascade of two affine tags; CBD, the chitin-binding domain; H6, the polyhistidine domain; HP, hybrid protein; IB, inclusion bodies; STI, soy trypsin inhibitor.
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Makarov, D.A., Zinchenko, A.A., Stepanenko, V.N. et al. Development of a Pilot Technology for the Production of the Recombinant Human Enteropeptidase Light Chain in Soluble and Immobilized Forms. Russ J Bioorg Chem 46, 1052–1060 (2020). https://doi.org/10.1134/S1068162020050143
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DOI: https://doi.org/10.1134/S1068162020050143