No abstract

The natural ability of many proteins to polymerize into highly structured filaments has been harnessed as scaffolds to align functional molecules in a diverse range of biomaterials. Protein-engineering methodologies also enable the structural and physical properties of filaments to be tailored for specific biomaterial applications through genetic engineering or filaments built from the ground up using advances in the computational prediction of protein folding and assembly. Using these approaches, protein filament-based biomaterials have been engineered to accelerate enzymatic catalysis, provide routes for the biomineralization of inorganic materials, facilitate energy production and transfer, and provide support for mammalian cells for tissue engineering. In this review, we describe how the unique structural and functional diversity in natural and computationally designed protein filaments can be harnessed in biomaterials. In addition, we detail applications of these protein assemblies as material scaffolds with a particular emphasis on applications that exploit unique properties of specific filaments. Through the diversity of protein filaments, the biomaterial engineer's toolbox contains many modular protein filaments that will likely be incorporated as the main structural component of future biomaterials.

Background 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. 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. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Results 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. We employed the ultrastable filamentous protein gamma-prefoldin as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. 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. 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. Subsequently, functionalized protein filaments were purified from bacterial lysates using MMC and shown to be free of unincorporated subunits. The assembly and purification of protein filaments with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. Finally, we compared our MMC workflow to anion exchange chromatography with the purification of encapsulin nanocompartments containing a fluorescent protein as a cargo, demonstrating the versatility of the protocol and that the purity of the assembly is comparable to more traditional procedures. Conclusions We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices. Graphic Abstract

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. 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. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Here, we developed an efficient approach to purify functionalized protein filament assemblies directly from bacterial lysates in a single step through a type of multimodal chromatography that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. In this approach, the ultrastable filamentous protein gamma-prefoldin was employed as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using MMC was optimized across a wide range of salt concentrations and pH. Subsequently, functionalized protein assemblies were purified from bacterial lysates using MMC in a single step and shown to befree of unincorporated subunits. The assembly and purification of protein nanostructures with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Förster resonance energy transfer, and transmission electron microscopy. We envision that the use of MMC will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.