Immobilization of the restriction endonuclease EcoRI - Usefulness of a polyclonal antibody support

The restriction enzyme EcoRI has been immobilized by using an immunoaffinit y-based procedure on Sepharose 4B. The antibody support, prepared by coupli ng the gamma-globulin fraction of serum of immunized rabbits to CNBr-activa ted Sepharose-4B, was highly effective in binding EcoRI from solution altho ugh only about half of the bound activity appeared to be expressed by the i mmobilized preparations. Restriction activity of EcoRI immobilized on the a ntibody support was indistinguishable from that of soluble enzyme on the li near lambda-DNA and supercoiled plasmids pBR322 and pBHLUC P. Binding to th e antibody support markedly enhanced the resistance of EcoRI to heat inacti vation, and the preparation, unlike the native enzyme, retained significant activity after exposure to a temperature of 65 degrees C. It was also poss ible to immobilize EcoRI directly from the cell lysates of Escherichia coli pMB4, an EcoRI overproducing strain. The immobilized preparation did not p ossess nonspecific nuclease activity that was prominent in the lysates, sug gesting specificity in the binding of EcoRI to the antibody support. (C) 19 99 Elsevier Science Inc. All rights reserved.