Democratizing the rapid screening of protein expression for materials development

Morris, Melody; Bataglioli, Rogério Aparecido; Danielle, J.; Yang, Yun Jung; Paloni, Justin M.; Mills, Carolyn E.; Schmitz, Zachary D.; Ding, Erika A.; Huske, Allison C.; Olsen, Bradley D.

doi:10.1039/d2me00150k

Cited by 6 publications

(7 citation statements)

References 69 publications

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“…For troubleshooting this protocol, especially when studying large sets of similar enzymes, we recommend performing this type of purification analysis on a smaller test set before committing to the construction of a library in a particular construct or strain. While the SUMO tag has been used successfully on a variety of proteins 36 , 57 , 58 , this protocol would be amenable to other constructs that confer an affinity tag 59 and protease cleavage site 60 and different E. coli strains can also influence expression efficiency 61 .…”

Section: Resultsmentioning

confidence: 99%

“…Screening through these conditions is another opportunity where this high-throughput platform could be applied via the small-scale and automated testing of constructs, strains, and conditions to optimize expression efficiency. Recently, an automated protocol utilizing an OT-2 was reported for testing various vectors and E. coli expression strains 61 . Employing this strategy to optimize expression efficiency, followed by high-throughput purification as described in this manuscript, may help reduce protein production as a bottleneck in enzyme engineering.…”

Section: Discussionmentioning

confidence: 99%

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Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Norton-Baker,

Denton,

Murphy

et al. 2024

Sci Rep

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As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Norton-Baker,

Denton,

Murphy

et al. 2024

Sci Rep

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show abstract

“…https://doi.org/10.26434/chemrxiv-2024-wk4bn-v2 ORCID: https://orcid.org/0000-0001-5447-2845 Content not peer-reviewed by ChemRxiv. License: CC BY-NC-ND 4.0 Block V and the 12 variants were produced using directional cloning and recombinant protein expression in Escherichia coli (32), which was tolerant to all sequence mutations. Expressed proteins were isolated using immobilized metal affinity chromatography to capture 6×His-tagged proteins of interest, dialysis to remove excess ions, and lyophilization to remove water.…”

Section: Resultsmentioning

confidence: 99%

Calcium-responsive order-to-order transitions by repetitive bacterial proteins

Chang,

Huang,

Shambharkar

et al. 2024

Preprint

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Protein sequences dictate their functions, and sequence repetition is a common strategy to amplify function within modular, protein-based biomaterials and biotechnologies. Repetitive bacterial proteins have drawn interest for their calcium-responsive folding behavior, which stems from tandem repeats of the nonapeptide GGXGXDXUX in which X can be any amino acid and U is a hydrophobic amino acid. To determine the functional range of this nonapeptide, we modified a bacterial protein that forms β-roll structures in the presence of calcium. Sequence modifications focused on the repetitive region, including either global substitution of nonconserved residues or complete replacement with tandem repeats of the consensus nonapeptide GGAGXDTLY. Some sequence modifications disrupted the disorder-to-order transition associated with β-roll formation, despite conservation of the underlying nonapeptide sequence. Proteins enriched in smaller, hydrophobic amino acids adopted secondary structures in the absence of calcium and underwent order-to-order structural transitions in calcium-rich environments. In contrast, proteins with bulkier, hydrophilic amino acids maintained intrinsic disorder in the absence of calcium. These results indicate a significant role of nonconserved residues in calcium-responsive folding, thereby revealing a strategy to leverage these residues in the design of tunable, calcium-responsive biomaterials.

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“…Recent advances in de novo protein design have also enabled development of self-associating proteins not seen in nature, opening up new possibilities in the design of genetically encoded protein biopolymer networks (53). The recent emphasis in biotech on protein-based therapeutics promises to develop more efficient and costeffective ways to screen protein expression conditions and achieve large scale protein expression, which will further enable the usage of such recombinant protein materials (54,55).…”

Section: Discussionmentioning

confidence: 99%

PhoCoil: An Injectable and Photodegradable Single-component Recombinant Protein Hydrogel for Localized Therapeutic Cell Delivery

Gregorio,

DeForest

2024

Preprint

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Hydrogel biomaterials offer great promise for 3D cell culture and therapeutic delivery. Despite many successes, challenges persist in that gels formed from natural proteins are only marginally tunable while those derived from synthetic polymers lack intrinsic bioinstructivity. Towards the creation of biomaterials with both excellent biocompatibility and customizability, recombinant protein-based hydrogels have emerged as molecularly defined and user-programmable platforms that mimic the proteinaceous nature of the extracellular matrix. Here, we introduce PhoCoil, a dynamically tunable recombinant hydrogel formed from a single protein component with unique multi-stimuli responsiveness. Physical crosslinking through coiled-coil interactions promotes rapid shear-thinning and self-healing behavior, rendering the gel injectable, while an included photodegradable motif affords on-demand network dissolution via visible light. PhoCoil gel photodegradation can be spatiotemporally and lithographically controlled in a dose-dependent manner, through complex tissue, and without harm to encapsulated cells. We anticipate that PhoCoil will enable new applications in tissue engineering and regenerative medicine.

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Democratizing the rapid screening of protein expression for materials development

Abstract: Low-cost, high-throughput methods for the determination of high-yield protein expression conditions are developed and verified, to enable the rapid development of new protein materials, such as biosensors and biomaterials.

Cited by 6 publications

References 69 publications

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Calcium-responsive order-to-order transitions by repetitive bacterial proteins

PhoCoil: An Injectable and Photodegradable Single-component Recombinant Protein Hydrogel for Localized Therapeutic Cell Delivery

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