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Preparation of protease free and rnase free pancreatic dnase ec 3.1.4.5






Journal of Biological Chemistry 253(20): 7216-7219

Preparation of protease free and rnase free pancreatic dnase ec 3.1.4.5

When pancreatic DNase I[EC 3.1.4.5] is used as a specific biochemical reagent in the preparation of nuclear RNA or nuclear proteins, freedom from contaminating RNases or proteases is an important property of the enzyme preparation. A simple 1-step procedure was developed to effect complete removal of trypsin, chymotrypsin and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose (a column (0.9 .times. 60 cm) operated in series with a regeneratable 1 ml bed). Commercial preparations of DNase (about 10 mg) give a quantitative yield of the enzyme that is protease-free as evidenced by full stability for more than 10 days at pH 8 and 37.degree. C. Removal of the last traces of RNase was accomplished by affinity chromatography on a column (0.4 .times. 72 cm) of 5'-(4-aminophenyl-phosphoryl)-uridine 2'(3')-phosphate-Sepharose; the product is a highly active DNase that gives no detectable hydrolysis of RNA by assay on radioactive substrates.


Accession: 006173720



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