High‐throughput cell‐free screening of eukaryotic membrane protein expression in lipidic mimetics

Bruni, Renato; Laguerre, Aisha; Kaminska, Anna‐Maria; McSweeney, Seán; Hendrickson, Wayne A.; Liu, Qun

doi:10.1002/pro.4259

Cited by 8 publications

(15 citation statements)

References 78 publications

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“…All of these six targets were, however, expressed and soluble in Brij-35 (Figure 5A and data not shown). Overall, Brij-35 performed much better than DDM, solubilizing almost twice as many targets (29 vs. 15; see Bruni et al, 2021).…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…By using several lipidic mimetics from different categories (i.e., two detergents, two liposomes, two nanodiscs), a large percentage of targets tested can be solubilized. We recently reported on a screening of 61 eukaryotic targets and were able to show solubilization of 35 for a coverage of 57%, which is significantly higher than typically found in cell‐based expression membrane proteins in E. coli (Bruni & Kloss, 2013; Bruni et al., 2021; R. Bruni & B. Kloss, unpublished data). The balance of the unsuccessful targets falls in two classes: (a) those that did not give a product at all, that is, were negative when assaying the total reaction synthesis, and (b) targets that were synthesized but failed to solubilize, that is, were positive in the total reaction but the product was absent in the solubilized fraction.…”

Section: Commentarymentioning

confidence: 89%

“…However, insertion into liposomes is significantly less efficient than solubilization in detergent. Our own data suggest that insertion into liposomes is ∼30% as successful as detergent solubilization (Bruni et al, 2021). Still, there are several ways to improve the odds of success.…”

Section: Lipidic Mimeticsmentioning

confidence: 93%

“…To allow high-throughput screening, we use 96-well microdialysis deep-well blocks that are fitted with cartridges (3.5-or 10-kD cutoff) that can hold up to 110 μl of reaction mixture (see Materials and Figure 4). The surrounding well is then filled up with a ∼17fold excess volume of feeding mixture (Bruni et al, 2021). When including additives, a crucial consideration is whether to add them to both RM and FM or to the RM only.…”

Section: Brunimentioning

confidence: 99%

“…Detergents are arguably the most straightforward lipidic mimetic to set up in cell-free reactions. In our experience, the family of Brij detergents (polyoxyethylene alkyl-ethers), in particular Brij-35, have consistently given the best results for target solubilization (Bruni et al, 2021). Because Brij compounds are mild detergents, they can be used at high concentrations, which will facilitate protein solubilization.…”

Section: Lipidic Mimeticsmentioning

confidence: 99%

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High‐Throughput Cell‐Free Screening of Eukaryotic Membrane Proteins in Lipidic Mimetics

Bruni

2022

Current Protocols

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Membrane proteins (MPs) carry out important functions in the metabolism of cells, such as the detection of extracellular activities and the transport of small molecules across the plasma and organelle membranes. Expression of MPs for biochemical, biophysical, and structural analysis is in most cases achieved by overexpression of the desired target in an appropriate host, such as a bacterium. However, overexpression of MPs is usually toxic to the host cells and can lead to aggregation of target protein and to resistance to detergent extraction. An alternative to cell‐based MP expression is cell‐free (CF), or in vitro, expression. CF expression of MPs has several advantages over cell‐based methods, including lack of toxicity issues, no requirement for detergent extraction, and direct incorporation of target proteins in various lipidic mimetics. This article describes a high‐throughput method for the expression and purification of eukaryotic membrane proteins used in the author's lab. Basic Protocol 1 describes the selection and cloning of target genes into appropriate vectors for CF expression. Basic Protocol 2 describes the assembly of CF reactions for high‐throughput screening. Basic Protocol 3 outlines methods for purification and detection of target proteins. Support Protocols 1‐6 describe various accessory procedures: amplification of target, treatment of vectors to prepare them for ligation‐independent cloning, and the preparation of S30 extract, T7 RNA polymerase, liposomes, and nanodiscs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Target selection, construct design, and cloning into pET‐based expression vectors Support Protocol 1: Amplification of target DNA Support Protocol 2: Preparation of ligation‐independent cloning (LIC)‐compatible vectors Basic Protocol 2: Assembly of small‐scale cell‐free reactions for high‐throughput screening Support Protocol 3: Preparation of Escherichia coli S30 extract Support Protocol 4: Preparation of T7 RNA polymerase Support Protocol 5: Preparation of liposomes Support Protocol 6: Preparation of nanodiscs Basic Protocol 3: Purification and detection of cell‐free reaction products

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Section: Anticipated Resultsmentioning

confidence: 99%

Section: Commentarymentioning

confidence: 89%

Section: Lipidic Mimeticsmentioning

confidence: 93%

Section: Brunimentioning

confidence: 99%

Section: Lipidic Mimeticsmentioning

confidence: 99%

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High‐Throughput Cell‐Free Screening of Eukaryotic Membrane Proteins in Lipidic Mimetics

Bruni

2022

Current Protocols

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Opportunities to accelerate extracellular vesicle research with cell‐free synthetic biology

Kelwick

Webb

Heliot

et al. 2023

J of Extracellular Bio

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Extracellular vesicles (EVs) are lipid‐membrane nanoparticles that are shed or secreted by many different cell types. The EV research community has rapidly expanded in recent years and is leading efforts to deepen our understanding of EV biological functions in human physiology and pathology. These insights are also providing a foundation on which future EV‐based diagnostics and therapeutics are poised to positively impact human health. However, current limitations in our understanding of EV heterogeneity, cargo loading mechanisms and the nascent development of EV metrology are all areas that have been identified as important scientific challenges. The field of synthetic biology is also contending with the challenge of understanding biological complexity as it seeks to combine multidisciplinary scientific knowledge with engineering principles, to build useful and robust biotechnologies in a responsible manner. Within this context, cell‐free systems have emerged as a powerful suite of in vitro biotechnologies that can be employed to interrogate fundamental biological mechanisms, including the study of aspects of EV biogenesis, or to act as a platform technology for medical biosensors and therapeutic biomanufacturing. Cell‐free gene expression (CFE) systems also enable in vitro protein production, including membrane proteins, and could conceivably be exploited to rationally engineer, or manufacture, EVs loaded with bespoke molecular cargoes for use in foundational or translational EV research. Our pilot data herein, also demonstrates the feasibility of cell‐free EV engineering. In this perspective, we discuss the opportunities and challenges for accelerating EV research and healthcare applications with cell‐free synthetic biology.

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