Preparation of immobilized monomeric actin and its use in the isolation of protease-free and ribonuclease-free pancreatic deoxyribonuclease I.
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.
Nefsky
B
B
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
Bretscher
A
A
D000199:Actins; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D003850:Deoxyribonuclease I / Q000302:isolation & purification*; D008840:Microfilament Proteins / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000032:analysis; D011485:Protein Binding; D012260:Ribonucleases / Q000032:analysis
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C026615:villin; D012260:Ribonucleases; D003850:Deoxyribonuclease I; D010447:Peptide Hydrolases