In Vitro Expressed GPCR Inserted in Polymersome Membranes for Ligand‐Binding Studies

May, Sylvia; Andreasson-Ochsner, Mirjam; Fu, Zhikang; Low, Ying Xiu; Tan, Chin Hon; Hoog, Hans‐Peter M. de; Ritz, Sandra; Nallani, Madhavan; Sinner, Eva‐Kathrin

doi:10.1002/anie.201204645

Cited by 43 publications

(50 citation statements)

References 26 publications

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“…Later, selective deposition and self‐assembly of triblock copolymers (polymethyloxazoline‐polydimethylsiloxane‐polymethyloxazoline [PMOXA‐PDMS‐PMOXA], (ABA)) were demonstrated for fabrication of matrix arrays for dopamine receptor 2 (DRD2) membrane protein expression, producing membrane protein array for diagnostics or drug discovery purposes (Andreasson‐Ochsner et al, 2012). Likewise, production and integration of DRD2 into PMOXA (20) –PDMS (54) –PMOXA (20) and PBd (22) –PEO (13) block copolymer vesicles are used for ligand‐ and antibody‐binding assays (Figure 7e; May et al, 2013). Very recently, diblock copolymers of poly (ethylene oxide)‐b‐poly(butadiene) PEO‐b‐PBD is exploited to enhance folding of a mechanosensitive channel of large conductance membrane protein during cell‐free expression (Jacobs, Boyd, & Kamat, 2019).…”

Section: Cfps: Latest Platforms and Applications In Biotechnology Andmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

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Section: Cfps: Latest Platforms and Applications In Biotechnology Andmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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“…Incorporation of GPCRs into polymersomes via cell free expression has been previously demonstrated. In 2013, May et al incorporated the dopamine receptor D 2 (DRD2) into polymeric vesicles . However, they only observed ligand binding on nanoscale vesicles and were not able to show functionality since the G proteins were not present.…”

Section: ‐Ht1ar In Pgup Response To Changes In Antagonist Species Anmentioning

confidence: 99%

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality

Gutierrez

Jalali-Yazdi

Peruzzi

et al. 2016

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We successfully incorporated the human serotonin receptor, G protein coupled receptor (GPCR), 5-HT1AR, in micron scale polymeric giant unilamellar protein-vesicles (pGUPs). By utilizing an agarose rehydration technique for protein incorporation, the GPCR is inserted in biased orientation with the C-terminus cytosolic and N-terminus extracellular as found in the cell plasma membrane. The GPCR is fully functional within the polymeric bilayer exhibiting responses to various ligands. The entire population of incorporated GPCRs displaying activity in pGUPs remains fully functional after lyophilization for 120 hours.

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“…Alternatively, reconstitution of membrane proteins into lipid membranes results in a random distribution and reduced accessibility to ligands [14,15]. Currently, cell-free approaches showed the expression and co-translational insertion of GPCRs directly into a lipid bilayer [16,17], in "nanodiscs" which are discoidal phospholipid bilayers stabilized by amphiphilic helical membrane proteins [18] or even in amphiphilic block-copolymer membranes [19]. In terms of the cotranslational insertion into lipid bilayers or liposomes, a unidirectional orientation of the GPCR molecules in the membrane structure is observed, since the protein synthesis machinery is added to the exposed plane of the bilayer membrane surface [20].…”

Section: Introductionmentioning

confidence: 99%

Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs)

Ritz¹,

Hulko²,

Zerfaß³

et al. 2013

Biochimie

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Although the identification of the multigene family encoding mammalian olfactory receptors were identified more than 20 years ago, we are far from understanding olfactory perception because of the difficulties in functional expression of these receptors in heterologous cell systems. Cell-free (CF) or in vitro expression systems offer an elegant alternative route to cell based protein expression, as the functional expression of membrane proteins can be directly achieved from the genetic template without the need of cell cultivation and protein isolation. Here we investigated in detail the cell-free expression and membrane insertion of the olfactory receptor OR5 in dependence of different experimental conditions like probing different origins of the cell-free expression system (from bacteria, via plants and insects toward mammalian system) and lipid composition of the respective extracts. We provided substantial biochemical indications by radioactive labeling based on [(35)S]-methionine, followed by proteolytic digestion, and we found that the insertion of the olfactory receptor OR5 into liposomes resulted in an unidirectional orientation with the binding side exposed into the aqueous space, resembling the native orientation in the cilia of the olfactory neurons. We report the different results in synthesis capacity for the different in vitro systems employed as we like to demonstrate the first in vitro kit toward and ex situ and ex vivo odorant receptor array.

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In Vitro Expressed GPCR Inserted in Polymersome Membranes for Ligand‐Binding Studies

Cited by 43 publications

References 26 publications

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality

Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs)

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