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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/42900
Title: High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli
Authors: Pornpimol Mahamad
Chuenchit Boonchird
Watanalai Panbangred
Mahidol University
Keywords: Biochemistry, Genetics and Molecular Biology;Immunology and Microbiology
Issue Date: 1-Jul-2016
Citation: Applied Microbiology and Biotechnology. Vol.100, No.14 (2016), 6319-6330
Abstract: © 2016, Springer-Verlag Berlin Heidelberg. 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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84961675188&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/42900
ISSN: 14320614
01757598
Appears in Collections:Scopus 2016-2017

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