Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Yeliseev, Alexei; Berg, Arjen van den; Zoubak, Lioudmila; Hines, Kirk G.; Stepnowski, Sam; Williston, Kyle; Wang, Yan; Gawrisch, Klaus; Zmuda, Jonathan F.

doi:10.1038/s41598-020-73813-7

Cited by 11 publications

(17 citation statements)

References 51 publications

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“…To assess the possible role of post-translational modifications of CB 2 on its activation by the MRI ligands, the recombinant CB 2 receptor was isolated from two different expression cell lines, E. coli BL21 (DE3) 41 and Expi293F GNTI - 42 . The protein was purified and reconstituted into lipid bilayers containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) at a molar ratio of 3/1, either supplemented or not supplemented with cholesterol, as described in “ Methods ” 10 , 13 .…”

Section: Resultsmentioning

confidence: 99%

“…For comparison, the activation behavior of CB 2 isolated from the Expi293F GNTI - cells 42 and reconstituted into liposomes was studied (Fig. 7 e).…”

Section: Resultsmentioning

confidence: 99%

“…Expi293F HEK cells were obtained from ThermoFisher Scientific (Cat. No: A14527), and CB 2 expressed and membranes obtained in house as described elsewhere 42 . The E. coli cells BL21 (DE3) were obtained from EMD Millipore-Sigma (Cat.…”

Section: Methodsmentioning

confidence: 99%

“…In www.nature.com/scientificreports/ all cholesterol-containing liposomes, including those containing the brain lipid extract, the MRI-2646 ligand acted as a partial inverse agonist of the receptor. For comparison, the activation behavior of CB 2 isolated from the Expi293F GNTIcells 42 and reconstituted into liposomes was studied (Fig. 7e).…”

Section: Liposome-reconstituted Cb 2 and Post-translational Modificatmentioning

confidence: 99%

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Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Yeliseev

Iyer

Joseph

et al. 2021

Sci Rep

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Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.

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Section: Resultsmentioning

confidence: 99%

“…For comparison, the activation behavior of CB 2 isolated from the Expi293F GNTI - cells 42 and reconstituted into liposomes was studied (Fig. 7 e).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Liposome-reconstituted Cb 2 and Post-translational Modificatmentioning

confidence: 99%

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Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Yeliseev

Iyer

Joseph

et al. 2021

Sci Rep

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“…However, despite significant efforts over the past two decades, there has been limited success in expressing correctly folded and functionally active GPCR in CFPS systems 25 . An improved methodology has been described for functional E. coli expression of the class A rhodopsin-like GPCR human cannabinoid receptor type II (CB2), as well as from mammalian HEK293 and CHO cell cultures [26][27][28] . However, no successful CFPS of the CB2 receptor has been reported until now.…”

Section: N-linked Glycosylation Of Glycoproteins Expressed In Bylmentioning

confidence: 99%

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2022

Preprint

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Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 uL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

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Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Gupta¹,

Flaskamp²,

Roentgen³

et al. 2023

Biotech & Bioengineering

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Eukaryotic cell‐free protein synthesis (CFPS) can accelerate expression and high‐throughput analysis of complex proteins with functionally relevant post‐translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing.Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY‐2 cell culture (BY‐2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N‐glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale‐up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre‐scale reaction in a rocking‐type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields.Production of multimeric virus‐like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult‐to‐express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS‐CoV‐2 receptor‐binding domain; a human growth factor; and a G protein‐coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in‐depth PTM characterization of purified proteins through disulfide bond and N‐glycan analysis.Taken together, BYL is a promising end‐to‐end R&D to manufacturing platform with the potential to significantly reduce the time‐to‐market for high value proteins and biologics.

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Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Cited by 11 publications

References 51 publications

Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Cholesterol as a modulator of cannabinoid receptor CB2 signaling

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

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Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture

Cited by 11 publications

References 51 publications

Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Cholesterol as a modulator of cannabinoid receptor CB2 signaling

ALiCE®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Scaling eukaryotic cell‐free protein synthesis achieved with the versatile and high‐yielding tobacco BY‐2 cell lysate

Contact Info

Product

Resources

About

ALiCE^®: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system