FIELD: biotechnologies.
SUBSTANCE: invention refers to biotechnology and gene engineering and represents recombinant pHisTevTSIB0821 plasmid for expression in Escherichia coli cells of TSIB_0821 prolidase from Thermococcus sibiricus archean. The proposed plasmid includes NdeI/SalI-fragment of pET-22b(+) (Novagen) plasmid and a DNA fragment with the size of 1196 pairs of bases, which contains a fused gene consisting of the following structural elements: nucleotide sequence coding 6-histidine tag, nucleotide sequence coding the site of recognition/decomposition of TEV protease, and nucleotide sequence of TSIB_0821 gene, which are connected so that at their biosynthesis in E. coli cells a continuous reading frame can be maintained. E. coli Rosetta(DE3) strain is obtained, which is transformed with the above plasmid, - a producer of chimeric protein including amino acid sequence of TSIB_0821 prolidase, fused on N-end with the 6-histidine tag and the site of recognition/decomposition of TEV protease. A growth and induction method of a producer strain and a method for separation and cleaning from the obtained biomass of functionally active recombinant TSIB_0821 prolidase including the following technological process: two metal affine chromatographies, gel filtration, TEV protease treatment, dialysis, and concentration, have been developed.
EFFECT: invention allows obtaining recombinant prolidase that is similar as much as possible as to structure to its natural equivalent with high and stable yield, level of cleaning and functional activity.
3 cl, 3 ex
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Year |
Author |
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