The Activation of Mutant β-Galactosidase by Specific Antibodies

An immunosorbent was prepared of antiserum against purified wild type β-galactosidase coupled to Sepharose. Extracts of wild type Escherichia coli K12 3300 cells and of eleven mutant cells producing proteins activatable by specific antibodies were adsorbed to this immunosorbent. The adsorbed proteins could be eluted from the immunosorbent by 8 M urea. By co-electrophoresis in polyacrylamide gels containing 8 M urea, 0.01 M mercaptoethanol and 1% sodium dodecylsulfate in a buffer at pH 7.2 subunits of all the eleven [3]leucine-labelled mutant proteins were found to have the same size (molecular weight 130000) within the accuracy of the method (± 15000) as subunits of [14C]leucine-labelled wild type enzyme. After renaturation from urea solutions the molecular weights of the native forms of the eleven [3H]leucine labelled mutant proteins were compared to that of the 14C-labelled native wild type enzyme by co-sedimentation in sucrose gradients. The native proteins were found to be identical in size as well (molecular weights 520000—540000). Thus, activation of these eleven mutant proteins by specific antibody occurs at the four subunit structure level. Such purification on immunosorbents represents a rapid, easy and generally applicable method to be used in systems with immunologically cross-reacting mutant proteins, where the wild type protein can be obtained in sufficiently pure form.