METHOD FOR PURIFYING A RECOMBINANT ENZYME PREPARATION OF PHOSPHOLIPASE A2 FROM A PRODUCER STRAIN PICHIA PASTORIS Russian patent published in 2021 - IPC C12N15/52 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. A method for the production and purification of the recombinant phospholipase A2 of Streptomyces violaceoruber from the producer strain Pichia pastoris is proposed. The invention allows to obtain recombinant secretory phospholipase A2 of Streptomyces violaceoruber with high concentration and purification degree of more than 90%. A method for purifying phospholipase A2 from a composition comprising phospholipase A2 and components of a cell culture medium, comprising concentrating the culture fluid on TAMI ceramic filters (10 kDa) with a 5-fold concentration of the culture fluid volume, characterized in that the method consists of several successive stages, after concentration, the culture broth concentrate is diluted in a 10-fold volume of 20 mM Tris-HCl pH = 8.0, after which the diluted culture broth solution is applied to an anion-exchange chromatographic column with quaternary amines attached to the carrier and eluted in 20 mM Tris-HCl pH = 8.0 gradient 0-1M sodium chloride, and sodium chloride is added to 2M to the fractions not bound to the anion-exchange chromatographic column, then the combined fractions with a carrier for chromatography of hydrophobic interactions containing butyl groups are applied to the chromatographic column, then the combined fractions are eluted in 20 mM Tris -HCl p Н = 8.0, in a sodium chloride gradient from 2M to zero, lowering the sodium chloride content, after which fractions in the range of 65-100% of the eluate containing highly active highly purified phospholipase A2 are collected, which are combined after hydrophobic interaction chromatography.

EFFECT: invention makes it possible to obtain a highly purified preparation of recombinant phospholipase A2 with a high (more than 90%) degree of purification with a yield of more than 50% of the initial protein content in the culture liquid.

1 cl, 3 ex