Molecular Biotechnology

, Volume 59, Issue 4–5, pp 117–127 | Cite as

Molecular Cloning, Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant Escherichia coli

  • Naphatsamon Uthailak
  • Pornpimol Mahamad
  • Pamorn Chittavanich
  • Somchai Yanarojana
  • Wassana Wijagkanalan
  • Jean Petre
  • Watanalai Panbangred
Original Paper

Abstract

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.

Keywords

Cross-reacting material 197 CRM197EK Escherichia coli Molecular chaperones Molecular modeling 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biotechnology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Mahidol University - Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU: CRC), Faculty of ScienceMahidol UniversityBangkokThailand
  3. 3.Department of Pharmacology, Faculty of ScienceMahidol UniversityBangkokThailand
  4. 4.BioNet-Asia Co., Ltd., 19 Udomsuk 37BangkokThailand

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