Subsequently, functionalized protein filaments were purified from bacterial lysates using MMC and shown to be free of unincorporated subunits. In addition, we show that pre-processing of the samples with tangential flow filtration to remove nucleotides and metabolites improves resin capacity, and that post-processing with Triton X-114 phase partitioning is useful to remove lipids and any remaining lipid-associated protein.
The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was tested across a wide range of salt concentrations and pH, demonstrating that the MMC resin is robust, however the optimal choice of salt species, salt concentration, and pH is likely dependent on the protein nanostructure to be purified. We employed the ultrastable filamentous protein gamma-prefoldin as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/Sp圜atcher conjugation chemistry. We developed an efficient approach to purify functionalized protein nanostructures directly from bacterial lysates through a type of multimodal chromatography (MMC) that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials.
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