CRM197 is the diphtheria toxin mutant used in many conjugate vaccines. A fusion CRM197 (fCRM197) containing all the tags conferred by the pET32a vector was produced as a soluble protein in Escherichia coli co-expressing several chaperone proteins in conjunction with low temperature cultivation. Trigger factor (Tf) enhanced formation of soluble fCRM197 (150.69 +/- 8.95 mu g/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 A degrees C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 A degrees C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 +/- 13.14 mu g/mL and 150.07 +/- 8.13 mu g/mL, respectively). Transmission electron microscopy (TEM) demonstrated that the formation of inclusion protein as well as cell lysis was reduced in cultures grown at 15 A degrees C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 A degrees C. The viability of Tf-expressing cells was enhanced when cultured at 15 A degrees C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (lambda DNA) at 37 A degrees C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 A degrees C (80.80 and 62.73 %, respectively) and at 15 A degrees C (7.34 and 24.79 %, respectively). These results demonstrating nuclease activity, enhanced cell lysis, and reduced cell viability are consistent with the finding of lower fCRM197 yield when cultivation and induction times were prolonged at 25 A degrees C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host.