Naphatsamon UthailakPornpimol MahamadPamorn ChittavanichSomchai YanarojanaWassana WijagkanalanJean PetreWatanalai PanbangredMahidol UniversityBioNet-Asia Co.2018-12-212019-03-142018-12-212019-03-142017-05-01Molecular Biotechnology. Vol.59, No.4-5 (2017), 117-12715590305107360852-s2.0-85015768759https://repository.li.mahidol.ac.th/handle/123456789/41911© 2017, Springer Science+Business Media New York. 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyChemical EngineeringImmunology and MicrobiologyArticleSCOPUS10.1007/s12033-017-0001-3