METHOD OF WHEAT CYSTEINE PROTEASE PROTEINS FAMILY (TRITICUM AESTIVUM) PRODUCING AND PREPARATION OF TRITIKAIE-ALPHA PROTEIN, OBTAINED USING SAID METHOD Russian patent published in 2016 - IPC C12N9/00 A61K38/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and represents method of tritikaine-alpha wheat truncated shape producing, having sequence of SEQ ID NO: 2 (shortTRIT-α), recombinant expressed in bacterial system, consisting in culturing of cells E.coli JM109, transformed by plasmid pQE80L_shortTRIT-α, containing DNA sequence coding shortTRIT-α protein, in LB medium with addition of ampicillin at 37 °C under aerobic conditions for 12-14 h, nutrient medium is inoculated with inoculum, culture is raised until optical density of A₆₀₀ 0.6-0.8, induced by 1 mM isopropylite-β-D-galactoside and raised for another 2.5-3 hours; target protein shortTRIT-α cleaning is carried out by affine metal-chelate chromatography: deposited by centrifugation expression culture cell biomass is re-suspended in buffer containing 0.01 M of Tris-HCl, pH 8.0 and homogenized on ultrasonic disintegrator for 1 min at 4 °C, produced after lysate centrifugation residue is washed with initial buffer and dissolved in buffer A, consisting of 6 M of guanidine-chloride and 0.05 M of Tris-HCl, pH 7.8, solution is clarified by centrifugation and applied on column with activated nickel ions iminodiacetate-sepharose, balanced by buffer A, sorbent is successively washed with balancing buffer A and same buffer containing 8 M of urea and 0.005 M of imidazole, protein is eluted with buffer A containing 8 M of urea and 0.25 M of imidazole, then eluate is added into cooled buffer of 0.05 M of Tris-HCl, 0.5 M of arginine-chloride, 2 M of urea, pH 7.8 at ratio of 1:5 and stirred for 1 h at 4 °C, solution is dialyzed against 0.05 M of Tris-HCl, pH 7.8 at 4 °C, supernatant produced after dialysate centrifugation, is concentrated on Amicon cell with RM-10 (Millipore) membrane, with subsequent dialysis against phosphate-salt buffer PBS, pH 7.4, at 4 °C, concentration of shortTRIT-α protein is determined, aliquoted by glass bottles, freezing and lyophilized.

EFFECT: invention enables to obtain pure protein preparation with tritikaine-alpha wheat activity with high degree of efficiency.

3 cl, 6 dwg, 4 ex