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Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1)

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Abstract

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1′ + S3′ specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

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Abbreviations

a.a.:

amino acid

c.f.:

culture fluid

Chn:

chymosin

CHO:

Chinese hamster ovary

CN:

casein

MCA:

milk-clotting activity

PA:

proteolytic activity

ProChn:

prochymosin

rChn-Cap:

recombinant roe deer chymosin

rChn-Bos:

recombinant cow chymosin

rChn-Cam:

recombinant one-humped camel chymosin

TS:

thermostability

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Funding

The study was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Agreement no. 075-15-2021-1355 dated October 12, 2021) as part of the implementation of certain activities of the Federal Scientific and Technical Program for the Development of Synchrotron and Neutron Research and Research Infrastructure for 2019-2027.

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Authors and Affiliations

  1. State Research Center of Virology and Biotechnology VECTOR, 630559, Koltsovo, Russia

    Denis E. Murashkin, Svetlana V. Belenkaya & Dmitrii N. Shcherbakov

  2. Novosibirsk State University, 630090, Novosibirsk, Russia

    Svetlana V. Belenkaya

  3. Genomics Core Facility, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

    Aleksandr A. Bondar

  4. Federal Altaic Scientific Center of Agrobiotechnology, 656910, Barnaul, Russia

    Vadim V. Elchaninov

  5. Altai State University, 656049, Barnaul, Russia

    Dmitrii N. Shcherbakov

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  1. Denis E. Murashkin
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  2. Svetlana V. Belenkaya
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  3. Aleksandr A. Bondar
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Murashkin and Belenkaya wrote the manuscript; Bondar created the figures; Elchaninov and Shcherbakov edited the text of the article.

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Correspondence to Svetlana V. Belenkaya.

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The authors declare no conflict of interest in financial or any other sphere. This article does not contain any studies with human participants or animals performed by any of the authors.

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Murashkin, D.E., Belenkaya, S.V., Bondar, A.A. et al. Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1). Biochemistry Moscow 88, 1284–1295 (2023). https://doi.org/10.1134/S0006297923090080

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