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Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
23803 43474 2011 8 PDF Available
Title
Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli
Abstract

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.

Keywords
CRM197, cross-reacting material 197; rCRM197, untagged recombinant cross-reacting material; rCRM197his, his-tagged recombinant cross-reacting material; HB-EGF, heparin binding epidermal growth factorCross-reacting material 197; Synthetic gene; Overexpress
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Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 156, Issue 4, 20 December 2011, Pages 245–252
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us