School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
Recombinant Products Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, Australia.
J Nanobiotechnology. 2023 Feb 24;21(1):66. doi: 10.1186/s12951-023-01817-2.
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.
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.
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.
通过单个亚基的自组装产生的蛋白质纳米结构是一种有吸引力的支架，可以用于附着和定位功能分子，应用于生物材料、代谢工程、组织工程和大量纳米材料。然而，多组分蛋白质纳米材料的组装通常是一个繁琐的过程，需要将每个蛋白质组件分别表达和纯化，然后再进行组装。此外，未纳入最终组装的多余组件必须从溶液中去除，因此需要额外的处理步骤。
我们开发了一种有效的方法，通过一种结合了大小排阻、亲水相互作用和离子交换的多模式色谱（MMC），直接从细菌裂解物中纯化功能化的蛋白质纳米结构，从而将重组蛋白组装体与多余的游离亚基和细菌蛋白分离。我们使用超稳定丝状蛋白γ-前折叠蛋白作为材料支架，通过 SpyTag/SpyCatcher 缀合化学可以将各种蛋白质结构域功能化。通过 MMC 从细菌裂解物中纯化重组γ-前折叠蛋白丝的实验测试了广泛的盐浓度和 pH 值范围，结果表明 MMC 树脂是稳健的，然而，最佳的盐种类、盐浓度和 pH 值选择可能取决于要纯化的蛋白质纳米结构。此外，我们还表明，用切向流过滤对样品进行预处理以去除核苷酸和代谢物可以提高树脂的容量，用 Triton X-114 相分离进行后处理有助于去除脂质和任何残留的与脂质相关的蛋白质。随后，使用 MMC 从细菌裂解物中纯化功能化的蛋白质丝，并证明其不含未掺入的亚基。使用聚丙烯酰胺凝胶电泳、荧光共振能量转移和透射电子显微镜证实了具有不同功能化程度的蛋白质丝的组装和纯化。最后，我们将我们的 MMC 工作流程与阴离子交换色谱法进行了比较，用于纯化含有荧光蛋白作为货物的封装蛋白纳米隔室，证明了该方案的多功能性，并且组装的纯度与更传统的方法相当。
我们设想，使用 MMC 将增加蛋白质纳米结构原型制作的吞吐量，并能够扩大蛋白质纳米器件的生物生产。
