RECOMBINANT PLASMID DNA PACYCLANS FOR TRANSFER AND EXPRESSION GENE ENCODING ERWINIA CAROTOVORA (ECAR-LANS) L-ASPARAGINASE IN ESCHERICHIA COLI CELLS AND METHOD FOR PREPARING RECOMBINANT ECAR-LANS FROM BIOMASS OF ESCHERICHIA COLI PRODUCER STRAINS

摘要

FIELD: genetic engineering, biotechnology, molecular biology. SUBSTANCE: invention can be used in medico-biological industry. Invention proposes the constructed recombinant plasmid (pACYCLANS) comprising the natural sequence of gene encoding L-asparaginase in Erwinia carotovora (ECAR-LANS) that is under control of promoter and terminator of phage T7 RNA-polymerase and encodes the full-scale enzyme precursor with signal peptide for export of protein to periplam followed by processing. Strain- producers with content of recombinant L-asparaginase 8-12% are prepared by transformation of strains E. coli with indicated plasmid that are able to synthesize phage T7 RNA-polymerase. For isolation and purification of enzyme from biomass method involves disruption of cells by ultrasonic disintegration, fractionation with ammonium sulfate and chromatography on column with SP-Sepharose FF. Invention provides preparing the purified L-asparaginase preparation with high yield. EFFECT: improved preparing method. 2 cl, 3 dwg, 1 tbl