High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

Abstract

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.

Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

“…This new version of the protein was named An2-CNF1-H8, as it still possessed a C-terminal polyhistidine tag (Figure 1A). The recombinant expressions of both CNF1-H8 and An2-CNF1-H8 were carried out at a low temperature of 15 • C, as previously reported [13]. The analysis of total cellular extracts obtained at the end of the bacterial growths indicated that an overexpression was clearly visible for both the constructs via SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 1(B1)) and western blot (Figure 1(B2)).…”

“…We previously reported an efficient strategy for the production and purification of the CNF1 toxin as a his-tagged recombinant protein (CNF1-H8) from E. coli extracts [13]. In the present manuscript, we describe the production and purification of the An2-CNF1-H8 variant, a CNF1 variant displaying an N-terminal Angiopep tag potentially endowed with the ability to allow BBB crossing.…”

“…It turned out that the amount of recombinant An2-CNF1-H8 variant was significantly higher, but it largely accumulated as inclusion bodies, although the production was carried out at 15 • C by using only 0.5 mM inducer. Lowering of the production temperature can be a successful approach to get the soluble product when the protein has the tendency to accumulate as inclusion bodies, as in the case of CNF1-H8 [13]. However, the higher production yields achieved for An2-CNF1-H8 shifted the equilibrium towards the formation of insoluble aggregates.…”

“…Conversely, E. coli synthesizing An2-CNF1-H8 suffered from the formation of inclusion bodies, distinguishable both in the Coomassie-stained gel and in western blot in the right part of Figure 1C. Despite a considerable loss of An2-CNF1-H8 as insoluble aggregates, its recovery in the cellular soluble fraction was still feasible using a previously established procedure [13].…”

“…We have previously demonstrated that a carboxy terminally his-tagged version of CNF1 (CNF1-H8) can be efficiently expressed in and purified from recombinant E. coli BL21(DE3) cells [13]. In this study, we attempted the production of a slightly modified version of the same toxin harboring an N-terminal peptide, named Angiopep-2 (An2), and capable of interacting with low-density lipoprotein receptor-related protein-1 (LRP1).…”