METHOD TO PRODUCE MODIFIED FORM OF TRYPTOPHANYL-tRNA-SYNTHETASE

摘要

FIELD: biotechnologies.SUBSTANCE: animal pancreas tissues are ground and extracted. The extract is separated from cake. Extrinsic nucleic acids are deposited and removed with the help of streptomycin sulfate. Admixtures are repeatedly deposited from supernatant by salting out with ammonium sulfate. Stagewise fractionating of the extract is carried out. At the first stage of fractionating the method of ion-exchange chromatography is used with application of DEAE-cellulose DE-52. Stepwise elution is carried out with a buffer solution containing 25 mM of tris-HCl at pH 7.5, 1mM of EDTA, 0.1 mM of 2-mercaptoethanol and stabiliser of modified form of TRNA-synthetase in the form of a complex of substrates of 0.2 mM of L-tryptophan and 1.0 mM of Mg-ATP. Elution is carried out until optical density value is achieved as not more than 0.1 p.u./ml at 280 nm. Extrinsic proteins are removed with a buffer solution containing 0.09M NHCl. The cleaned fraction is washed with a buffer solution containing 0.16M NHCl. The produced mixture is deposited with ammonium sulfate until 60% of saturation. The residue is centrifuged and dissolved with the buffer solution containing 0.1M NHCl. A protease inhibitor is added to the solution as 1 mM of phenyl methyl sulphonyl fluoride (PMSF) or 5 mM of disopropylfluorophosphate (DFP). The second stage of fractionating is carried out by the method of ion-exchange chromatography using DEAE-cellulose DE-52 with stepwise elution using a linear gradient of ammonium chloride salt 0.01 -0.06M. Fractions are extracted with the modified form of tryptophanyl-TRNA-synthetase (TRNA synthetase). The target product is concentrated by salting out with ammonium sulfate until 100% of saturation.EFFECT: invention makes it possible to increase yield of a modified form of tryptophanyl-TRNA-synthetase.2 cl, 2 dwg, 1 tbl, 10 ex