Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor

Li, Jianyong; Liu, Xing‐Ping; Man, Yahui; Chen, Qian; Pei, Di; Wu, Wenjian

doi:10.1016/j.pep.2019.03.001

Cited by 5 publications

(4 citation statements)

References 61 publications

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“…Another detergent that should be tested is n ‐dodecyl‐β‐D‐maltopyranoside (DDM). We and others have found it to perform adequately in cell‐free systems, albeit to a lesser degree than Brij‐35 (Bruni et al., 2021; Hein et al., 2014; J. Li et al., 2019). As with Brij‐35, a range of concentration should be used, that is, 0.02%‐1% (corresponding to ∼2×‐110× CMC).…”

Section: Commentarymentioning

confidence: 88%

“…As with Brij‐35, a range of concentration should be used, that is, 0.02%‐1% (corresponding to ∼2×‐110× CMC). However, many of the more conventional detergents used for extraction, such as octyl‐β‐D‐glucoside (OG) and phosphocholine (FCs), are too harsh for cell‐free experiments, most likely because they denature the translation machinery (Hein et al., 2014; J. Li et al., 2019). We have also found GDN, a synthetic version of digitonin, to work well (R. Bruni, unpublished data; GDN can be obtained from Anatrace).…”

Section: Commentarymentioning

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: Commentarymentioning

confidence: 88%

Section: Commentarymentioning

confidence: 99%

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

Bruni

2022

Current Protocols

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“…However, for getting the OR protein, these cells are lysed using sonication, and the insoluble fractions are collected, or handled by a membrane-protein-extraction kit to extract the functional protein. As for the cell-free synthesis, researchers only need to add the DNA template and reagents into the device, then it will automatically produce the target protein [114][115][116][117][118]. This method can avoid the issues such as protein aggregation and cytotoxicity which are usually encountered in heterologous expression.…”

Section: Or Protein-based Odor Biosensorsmentioning

confidence: 99%

Biosensors for Odor Detection: A Review

Deng,

Nakamoto

2023

Biosensors

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Animals can easily detect hundreds of thousands of odors in the environment with high sensitivity and selectivity. With the progress of biological olfactory research, scientists have extracted multiple biomaterials and integrated them with different transducers thus generating numerous biosensors. Those biosensors inherit the sensing ability of living organisms and present excellent detection performance. In this paper, we mainly introduce odor biosensors based on substances from animal olfactory systems. Several instances of organ/tissue-based, cell-based, and protein-based biosensors are described and compared. Furthermore, we list some other biological materials such as peptide, nanovesicle, enzyme, and aptamer that are also utilized in odor biosensors. In addition, we illustrate the further developments of odor biosensors.

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“…[ 80 ] In recent years, cell‐free protein expression systems have emerged, providing an alternative approach to address heterologous system expression. [ 81 ] It takes only a few hours to complete the integrated expression process. This system simply mimics the natural cytoplasmic environment, so it does not require living cells, and therefore it avoids toxic effects known from traditional cell‐based expression.…”

Section: Biorecognition Element Design and Selectionmentioning

confidence: 99%

Artificial Olfactory Biohybrid System: An Evolving Sense of Smell

Qin

Wang

et al. 2022

Advanced Science

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The olfactory system can detect and recognize tens of thousands of volatile organic compounds (VOCs) at low concentrations in complex environments. Bioelectronic nose (B-EN), which mimics olfactory systems, is becoming an emerging sensing technology for identifying VOCs with sensitivity and specificity. B-ENs integrate electronic sensors with bioreceptors and pattern recognition technologies to enable medical diagnosis, public security, environmental monitoring, and food safety. However, there is currently no commercially available B-EN on the market. Apart from the high selectivity and sensitivity necessary for volatile organic compound analysis, commercial B-ENs must overcome issues impacting sensor operation and other problems associated with odor localization. The emergence of nanotechnology has provided a novel research concept for addressing these problems. In this work, the structure and operational mechanisms of biomimetic olfactory systems are discussed, with an emphasis on the development and immobilization of materials. Various biosensor applications and current developments are reviewed. Challenges and opportunities for fulfilling the potential of artificial olfactory biohybrid systems in fundamental and practical research are investigated in greater depth.

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Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor

Cited by 5 publications

References 61 publications

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

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

Biosensors for Odor Detection: A Review

Artificial Olfactory Biohybrid System: An Evolving Sense of Smell

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