A.V. Solovyev –

Ph.D. (Biol.), Associate Professor, Leading Research Scientist, Laboratory of Molecular Biology, 

Research Center of Fundamental and Applied Problems of Bioecology and Biotechnology; 

Associate Professor, Department of Biology and Chemistry, Ulyanovsk State Pedagogical University, E-mail: solovyev_alexey@mail.ru

E.I. Antonova –

Dr. Sc. (Biol.), Professor, Department of Biology and Chemistry, Director of the Research Center  of Fundamental and Applied Problems of Bioecology and Biotechnology, Ulyanovsk State Pedagogical University E-mail: antonov_67@mail.ru

A.V. Khambikova –

Junior Research Scientist, Laboratory of Molecular Biology, Research Center of Fundamental  and Applied Problems of Bioecology and Biotechnology, Ulyanovsk State Pedagogical University E-mail: nastya010994@mail.ru

R.M. Khuzina –

Junior Research Scientist, Laboratory of Molecular Biology, Research Center of Fundamental  and Applied Problems of Bioecology and Biotechnology, Ulyanovsk State Pedagogical University E-mail: hr.apple@ya.ru

An artificial synthesis of DNA fragments that code target enzymes is necessary to obtain the producers of recombinant enzymes with optimized properties and improved technical characteristics of microbial synthesis due to optimization of codon frequencies and supplying of DNA with additional functional areas. The problem of direct phosphoramidite synthesis is concerned with quite low efficiency of synthesis of long DNA molecules (with length more than 150 bp).

Thus, the main aim of the present investigation is synthesis of cDNA of gene that codes chymosin of Bos taurus, based on optimized method of artificial DNA synthesis.

The chymosin (also known as rennin) is milk coagulant enzyme that is traditionally used in cheese making. The enzyme hydrolyzes the 105 (Phe) – 106 (Met) peptide bond of κ-casein. The recombinant chymosin in comparing with native one has several advantages that connected with economic expediency and possibility of optimization and improvement of properties by inserting of the changes in the gene structure.

As a result, three molecules of DNA with length 1193, 1155 and 1256 bp were synthesized. These molecules code enzyme chymosin and have optimized frequencies of codons and additional functional sequencies. The method of artificial DNA synthesis of DNA with length more than 1000 bp is optimized. The optimized method includes dividing of the scheme synthesis in two stages. Each of stages contains stages of elongation and amplification.

The results that were obtained will be subsequently used for development of recombinant producer of chymosin and microbial synthesis of the enzyme. The developed and approved technological map of artificial synthesis of chymosin gene can be used for synthesis of other genes with length more than 1000 bp.

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