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.
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.
Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.|Mahidol University - Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU: CRC), Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.
Naphatsamon
N
Uthailak
Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.|Mahidol University - Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU: CRC), Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.
Pornpimol
P
Mahamad
Department of Pharmacology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.
Pamorn
P
Chittavanich
Department of Pharmacology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.
Somchai
S
Yanarojana
BioNet-Asia Co., Ltd., 19 Udomsuk 37, Sukhumvit 103, Prakanong, Bangkok, 10260, Thailand.
Wassana
W
Wijagkanalan
BioNet-Asia Co., Ltd., 19 Udomsuk 37, Sukhumvit 103, Prakanong, Bangkok, 10260, Thailand.
Jean
J
Petre
Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand. Watanalai.pan@mahidol.ac.th.|Mahidol University - Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU: CRC), Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand. Watanalai.pan@mahidol.ac.th.
Watanalai
http://orcid.org/0000-0003-0141-9279
W
Panbangred
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D003198:Computer Simulation; D004926:Escherichia coli / Q000502:physiology*; D060847:Metabolic Engineering / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C028581:CRM197 (non-toxic variant of diphtheria toxin)
CRM197EK; Cross-reacting material 197; Escherichia coli; Molecular chaperones; Molecular modeling