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.
authors: Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, Bologna, Italy.
authors: Alessandra
authors: A
authors: Stefan
authors: Matteo
authors: M
authors: Conti
authors: Diego
authors: D
authors: Rubboli
authors: Lorenzo
authors: L
authors: Ravagli
authors: Enrica
authors: E
authors: Presta
authors: Alejandro
authors: A
authors: Hochkoeppler
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014508:Urea
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C028581:CRM197 (non-toxic variant of diphtheria toxin); D014508:Urea