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
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
