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

The structure of the Siberian roe deer (Capreolus pygargus) chymosin gene has been established for the first time and its exon/intron organization has been determined. The coding part of the C. pygargus chymosin gene was reconstructed and obtained as a DNA clone using the Golden Gate method. Comparative analysis of the sequences of prochymosins of roe deer, cow and single-humped camel revealed a number of amino acid substitutions in the sites forming the substrate-binding cavity of the enzyme and affecting the specificity subsites S4 and S1′ + S3′. The recombinant plasmid pIP1-Cap was constructed using the integration vector pIP1 for the expression of the roe deer prochymosin gene in CHO-K1 cells. A polyclone of CHO-K1-CYM-Cap cells was obtained, providing synthesis and secretion of recombinant prochymosin into the culture fluid of the producer. As a result of zymogen activation, a recombinant roe deer chymosin preparation with a total milk-clotting activity of 468.4 ± 11.1 IMCU/ml was obtained. The yield of recombinant roe deer chymosin was 500 mg/liter or ≈ 468,000 IMCU/liter, which exceeds the yield of genetically engineered chymosins in most of the expression systems used. The main biochemical properties of the obtained enzyme were compared with commercial preparations of recombinant chymosins of single-humped camel (Camelus dromedarius) and cow (Bos taurus). The specific milk-clotting activity of recombinant C. pygargus chymosin was 938 ± 22 IMCU/mg of protein and was comparable with the indicators of comparison enzymes. The nonspecific proteolytic activity of recombinant roe deer chymosin was 1.4-4.5 times higher than that of cow and camel enzymes. In terms of coagulation specificity, the recombinant C. pygargus chymosin occupied an intermediate position between the genetically engineered analogues of B. taurus and C. dromedarius chymosins. The threshold of thermal stability of recombinant roe deer chymosin was equal to 55°C. At 60°C, the enzyme retained <1% of the initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

About the authors

D. E Murashkin

State Research Center of Virology and Biotechnology ”Vector”

630559 Koltsovo, Russia

S. V Belenkaya

State Research Center of Virology and Biotechnology ”Vector”;Novosibirsk State University

Email: belenkaya.sveta@gmail.com
630559 Koltsovo, Russia;630090 Novosibirsk, Russia

A. A Bondar

Genomics Core Facility, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences

630090 Novosibirsk, Russia

V. V Elchaninov

Federal Altai Scientific Center of Agrobiotechnologies, Siberian Research Institute of cheese making

656910 Barnaul, Russia

D. N Shcherbakov

State Research Center of Virology and Biotechnology ”Vector”;Altai State University

630559 Koltsovo, Russia;656049 Barnaul, Russia

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