Preparative Production and Purification of Recombinant Human Cyclophilin A

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Anastasiia A. Kalinina¹, Alexander V. Kolesnikov², Arina V. Kozyr², Natalia L. Kulikova³, Maria A. Zamkova¹, Dmitry B. Kazansky¹, and Ludmila M. Khromykh^1,a*

¹N. N. Blokhin National Medical Research Center of Oncology, the Ministry of Health of the Russian Federation, 115478 Moscow, Russia

²State Research Center of Applied Microbiology and Biotechnology, 142279 Obolensk, Moscow Region, Russia

³Institute of Immunological Engineering, 142380 Lyubuchany, Moscow Region, Russia

^* To whom correspondence should be addressed.

Received January 26, 2022; Revised February 15, 2022; Accepted February 15, 2022

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In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.

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KEY WORDS: cyclophilin A, gene optimization, producer strain, isomerase activity, chemokine activity

DOI: 10.1134/S0006297922030063

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