title: High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli.
abstract: 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 μg/mL) to a greater degree than other chaperones when fCRM197 expression was induced at 25 °C for 12 h. However, prolonged cultivation resulted in a progressive reduction of fCRM197 accumulation. In contrast, at 15 °C cells, with or without Tf, fCRM197 accumulated to the highest level at 48 h (153.70 ± 13.14 μg/mL and 150.07 ± 8.13 μg/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 °C. Cell viability was substantially reduced in cells expressing Tf, compared to cultures without Tf, when fCRM197 was induced at 25 °C. The viability of Tf-expressing cells was enhanced when cultured at 15 °C. Both purified fCRM197 and CRM197 efficiently digested lambda DNA (λDNA) at 37 °C (92.78 and 97.45 %, respectively). Digestion efficiency of fCRM197 and CRM197 was reduced at 25 °C (80.80 and 62.73 %, respectively) and at 15 °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 °C. The present work provides a procedure for the high-level production of soluble fCRM197 using E. coli as a heterologous host.
authors.affiliation: 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.
authors.forename: Pornpimol
authors.initials: P
authors.lastname: Mahamad
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand.
authors.forename: Chuenchit
authors.initials: C
authors.lastname: Boonchird
authors.affiliation: 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.
authors.forename: Watanalai
authors.identifier: http://orcid.org/0000-0003-0141-9279
authors.initials: W
authors.lastname: Panbangred
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003080:Cold Temperature; D003470:Culture Media / Q000737:chemistry; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D050296:Microbial Viability; D046529:Microscopy, Electron, Transmission; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004167:Diphtheria Toxin; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C028581:CRM197 (non-toxic variant of diphtheria toxin)
keywords: CRM197; Chaperones; Escherichia coli; Expression