Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2

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Atefeh Khodakarami¹, Bahareh Dabirmanesh^1,a*, Sedigheh Asad², and Mohammad Khaledi¹

¹Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, 14115 Tehran, Iran

²Department of Biotechnology, College of Science, University of Tehran, 14155 Tehran, Iran

Received April 2, 2020; Revised September 22, 2020; Accepted October 13, 2020

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Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The K_m and k_cat values of 6.5 µM and 4.57 s^–1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

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KEY WORDS: carboxypeptidase G2, intein, methotrexate

DOI: 10.1134/S0006297921020073

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