Three immunization procedures were compared for the production of antibodies to the minor components of a complex E. coli protein (ECP) mixture: a conventional protocol and two methods that allow for the selective in vitro (cascade) or in vivo (passive) depletion of highly immunogenic proteins.An E. coli protein, designated ?, has been purified at least 1000-fold. Treatment of a eovalently closed DNA duplex containing negative superhelical turns with ? results in the loss of most of the superhelical turns. The loss of superhelical turns follows a gradual course rather than a one-hit mechanism. This reaction does not require a cofactor. No other change in the physical properties of the DNA could be detected. ? is capable of introducing a “swivel” reversibly into a DNA. A plausible mechanism is postulated.