Pronk S E, Hofstra H, Groendijk H, Kingma J, Swarte M B, Dorner F, Drenth J, Hol W G, Witholt B
J Biol Chem. 1985 Nov 5;260(25):13580-4.
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.
热不稳定肠毒素（LT）是从携带LT编码多拷贝质粒EWD299的大肠杆菌C600中大量（数克）且高纯度获得的。通过在一种能在稳定期早期实现高细胞密度的培养基上培养该菌株，与天然猪产肠毒素大肠杆菌相比，我们将每毫升LT的净产量提高了200倍。在可控孔玻璃上进行吸附和解吸附通常能在一个简单步骤中将LT纯化50至100倍，随后采用既定的纯化程序。LT具有形成大晶体的自然倾向，然而这些晶体是无序的。经过多次试验，找到了几乎消除无序的条件。通过使用氯化镉（CdCl2）和氟化钾（KF）作为共沉淀剂获得了质量好得多的晶体。通过对溶解晶体进行电泳判断，氯化镉产生的晶体不含A亚基。添加氟化钾会产生形状优美的晶体，其衍射超过2埃，适合进行高分辨率结构测定。
