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Abstract Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size‐ and charge‐based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size‐based separation coisolated protein contaminants, whereas size‐based TFF with charged‐based HPAEC dramatically improved purity of BEVs produced by probiotic Gram‐negativeEscherichia coliand Gram‐positive lactic acid bacteria (LAB).Escherichia coliBEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti‐inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large‐scale biomanufacturing of therapeutic BEV products.
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Efficient production of fluorophore‐labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase
Abstract Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti‐inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti‐chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion‐tagged chemokine is produced inEscherichia coli, then efficient cleavage of the N‐terminal fusion partner is carried out with lab‐produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab‐produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP‐fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti‐inflammatory therapeutic, showing a binding constant for vCCI:vMIP‐fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP‐fluor) can be used in competition assays with other chemokines and we report aKdfor vCCI:CCL17 of 14 μM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.
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- Award ID(s):
- 2112675
- PAR ID:
- 10527305
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Biopolymers
- Volume:
- 115
- Issue:
- 2
- ISSN:
- 0006-3525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/bip.23557
