Open AccessPreparation of immobilized monomeric actin and its use in the isolation of protease‐free and ribonuclease‐free pancreatic deoxyribonuclease I
Author(s) -
NEFSKY Bradley,
BRETSCHER Anthony
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14543.x
Subject(s) - deoxyribonuclease , deoxyribonuclease i , ribonuclease , biochemistry , monomer , formamide , pancreatic ribonuclease , bovine pancreatic ribonuclease , chemistry , affinity chromatography , biology , enzyme , dna , rna , polymer , organic chemistry , base sequence , gene
A procedure is described for the immobilization of monomeric actin so that about 30% of the immobilized protein is competent to bind the monomeric‐actin‐binding proteins bovine pancreatic deoxyribonuclease I and chicken villin. The intact tertiary structure of the immobilized actin is required to bind these proteins. Using this resin, a method has been developed for the affinity purification of pancreatic deoxyribonuclease I on a reusable actin column. It involves the binding of deoxyribonuclease I to immobilized actin, extensive washing of the column, followed by elution of the bound deoxyribonuclease I with 10 M formamide. After removal of the formamide, the deoxyribonuclease I has a higher specific activity than the starting material and contained no detectable protease or ribonuclease contamination. This preparation should find considerable application in molecular genetic studies where the enzyme is needed free of these particular contaminants. The affinity column should also be useful for the isolation of other, physiologically relevant, monomeric‐actin‐binding proteins.