A comparative study of chromatographic matrices for affinity chromatography of the diphtheria toxin variant Cross-Reacting-material 197

Cross-Reacting Material 197 (CRM197) is a variant of the diphtheria toxin characterised by a single mutation, a glycine-glutamic acid substitution at position 52 [1]. This mutation reduces its toxicity but maintains the same inflammatory and immunostimulant properties. The conventional industrial-scale production of CRM197 is performed using cultures of Corynebacterium diphtheriae, but recently we have proposed an alternative process for its over-expression in Escherichia coli [2]. Accordingly, the recombinant protein, bearing a short artificial histidine tag, was purified by a metal chelating affinity chromatography (IMAC), performed either under denaturing and native conditions. In order to investigate possible non-specific interactions between CRM197 and the matrix employed for the IMAC, we compared three types of matrices: a matrix consisting of agarose beads (GE Healthcare Life Sciences), a macroporous silica matrix (Machery-Nagel) and the Profinity IMAC resin, based on UNOsphere beads (Bio-Rad). The agarose-based matrix showed the higher non-specific sorption of the CRM197 protein, leading to a low final recovery. On the contrary, the Profinity IMAC resin proved the maximum yield, likely due to a low retention of the protein in the column via non-specific interactions with the matrix. Moreover, the Profinity resin was found to be suitable for both the purification and the refolding of CRM197 and, accordingly, the recovered protein featured enzymatic activity.