Heat-labile enterotoxin of Escherichia coli. Characterization of different crystal forms

Heat-labile enterotoxin (LT) was obtained in large quantities (several-gram amounts) and great purity from Escherichia coli C600 carrying the LT-coding multicopy plasmid EWD299. By growing this strain on a medium that allows high cell densities in the early stationary phase, we increased the net LT production per milliliter by a factor of 200, compared to natural porcine enterotoxigenic E. coli. Adsorption and redesorption on Controlled-Pore Glass usually resulted in a 50-100-fold purification of LT in one simple step, which was followed by established purification procedures. LT shows a natural tendency to form large crystals, which, however, are disordered. After numerous trials, conditions were found which virtually eliminated the disorder. Much better crystals were obtained by employing CdCl2 and KF as coprecipitating agents. CdCl2 yielded crystals which did not contain A subunits as judged by electrophoresis of dissolved crystals. Adding KF results in beautifully shaped crystals which diffracted beyond 2 A and are suitable for a high resolution structure determination.