CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However， it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33?±?17.47?μg/ml) under the optimal condition (induction with 0.1?mM IPTG at 20?°C for 24?h). Cells containing pG-Tf2， expressing trigger factor and GroEL-GroES， accumulated the highest amount of ... More
CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However， it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33?±?17.47?μg/ml) under the optimal condition (induction with 0.1?mM IPTG at 20?°C for 24?h). Cells containing pG-Tf2， expressing trigger factor and GroEL-GroES， accumulated the highest amount of soluble CRM197EKTrxHis at 111.24?±?10.40?μg/ml after induction for 24?h at 20?°C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37?°C with 8- and 4-h incubation， respectively. Molecular modeling of diphtheria toxin， CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding， consistent with the lack of toxicity. Therefore， CRM197EK might be used as a new potential carrier protein. However， further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.
