Recombinant expression and characterisation of the colicin n immunity protein

Some Escherichia coli express plasmid encoded toxins called colicins to eliminate ecological competitors. Colicins are a diverse group of toxins, usually divided into three groups: nucleases, pore-formers and peptidoglycan synthesis inhibitors. In addition, each colicinogenic plasmid codes for an immunity protein - a self-protection mechanism against its own toxin. Immunity proteins are highly specific to their cognate toxins but it is not clearly known how immunity proteins to pore-forming colicins achieve protection. This work focuses on the immunity protein for the smallest pore-forming colicin, colicin N. To investigate in vitro how the Colicin N immunity protein (CNI) neutralises the toxin, different overexpression and purification methods were tested. The fusion CNI-3C-HALO7-6His yielded the most protein, when expressed in C41 E. coli cells at 37 °C overnight in Terrific Broth. The fusion protein increases resistance to Colicin N dramatically and is therefore folded and localised correctly. Decyl β-Dmaltopyranoside is the most effective detergent to solubilise and stabilise the protein fusion. To investigate if CNI inactivates ColN by binding to it via hydrophobic α-helical interactions, the protein’s helical regions were defined in silico and swapped with the respective parts of the Colicin A immunity protein, CNI’s closest homologue, which provides immunity to Colicin A. The N-terminal region of CNI, including the first transmembrane helix, is not necessary for specific CNI-Colicin N interaction but the other helices are crucial for protein stability and function. CNI can be expressed in an active form fused to GFP for microscopy. Epifluorescent and TIRF microscopy revealed that CNI is localised to the cell periphery and appears to be very evenly distributed in the cell membrane. Colicin N addition does not affect CNI distribution. Single molecule tracking in TIRF microscopy showed that the diffusion rate of CNI does not change when ColN is added. Future work can pursue either of the three approaches used here to study CNI using in vitro structural analysis, site-directed mutagenesis of the active site or in vivo interaction of CNI with other E. coli inner membrane proteins.