Modification of bacterial microcompartments with target biomolecules via post-translational SpyTagging

Abstract

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational 'click' methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents.

Fig. 1. SpyTag peptides are typically incorporated through genetic encoding and recombinant protein expression. Previously, SpyTag modified proteins were produced recombinantly by the genetic modification of a host organism which produces the protein with the SpyTag attached. In this work, the post-translational modification of a protein, with a cyclooctyne, is used to modify the protein and allow the attachment of the SpyTag.

Fig. 2. SpyTag modification of citrine through a two-step labelling strategy. (A) Reaction scheme showing the non-specific lysine modification of citrine using BCN-OSu to give a click competent protein. This was then modified using 1st and 3rd generation SpyTags, synthesised by solid phase synthesis, which was modified with an azide group. (B) SDS-PAGE analysis of the reaction of citrine with BCN-OSu and then subsequent reactions with N₃ SpyTag001, 003 and also PEG-N₃. Samples which have been treated with PEG-N₃ are labelled as Y above the gel, and samples which do not have PEG-N₃ are labelled as N. The gel was Coomassie stained to show changes in electrophoretic mobility of each sample. (C) Deconvoluted electrospray MS spectra of citrine-BCN showing consecutive additions of BCN (+176 Da) to citrine.

Fig. 3. Post-translationally SpyTagged citrine (Citrine-SpyTag001/Citrine-SpyTag003) used to modify PduK-SpyCatcher fused empty Pdu bacterial microcompartments (SCeBMCs). (A) Reaction scheme showing the modification of BMCs using 1st and 3rd generation SpyTag conjugates through the PduK SpyCatcher. (B) SDS-PAGE analysis of the SpyCatcher BMC modification by SpyTag001 N₃ and SpyTag003 N₃. Shown are the SpyCatcher modified BMC (scBMC), the SpyTag modified citrine (st1citrine) and then 3 μL of SpyTag citrine treated with increasing amounts of scBMCs.

Fig. 4. TEM imaging of SpyCatcher BMCs modified with SpyTag Citrine by immunogold labelling. (A) SpyCatcher BMCs modified with the Citrine SpyTag1 protein. (B) SpyCatcher BMCs modified with the Citrine SpyTag3 protein. (C) Quantification of the number of citrine molecules on the BMC surface.