IMMUNOAFFINITY PURIFICATION OF DNA POLYMERASE-DELTA

A monoclonal antibody against human DNA polymerase delta (pol delta) w as isolated with properties suitable for its utilization for immunoaff inity chromatography. The antibody was immobilized after periodate oxi dation and coupled to a hydrazide-activated support. Starting from a p artially purified preparation, calf thymus pol delta was purified abou t 200-fold in a single step, Further purification on ssDNA-cellulose r esulted in isolation of a homogeneous preparation, The amount of enzym e isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 18-fold greater than can be achieved by conventional procedur es, This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield, The isolated enzyme consisted of two subunits of 125 and 50 kDa. Char acterization of the enzyme showed that these two subunits remained ass ociated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea. (C) 1995 Academic Press, Inc.