Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies

Krasnoselska, Ganna O.; Dumoux, Maud; Gamage, Nadisha; Cheruvara, Harish; Birch, J.R.; Quigley, Andrew; Owens, Raymond J.

doi:10.1007/978-1-0716-1406-8_5

Cited by 8 publications

(6 citation statements)

References 39 publications

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“…Transient gene expression in Expi293F cells was performed as described earlier [38]. As an alternative, the protocol described by the manufacturer of the Expi293 TM expression system can be used, which may result in higher transfection efficiencies.…”

Section: Transient Gene Expression In Mammalian Expi293f Cellsmentioning

confidence: 99%

“…The membrane fusion proteins were solubilized from the crude membrane fraction using 1% (v/v) DDM (n-dodecyl-β-D-maltopyranoside) [36,38] for 1 h on a vertically rotating platform in a cold room with a subsequent 1 h ultracentrifugation at 100,000× g (Optima L-80 XP Ultracentrifuge, Beckmann Coulter, Krefeld, Germany). The resulting supernatant was applied to either pre-equilibrated resins Ni-NTA ® -Sepharose 6 Fast Flow (Cytiva, Freiburg, Germany) for His6 constructs and Strep-Tactin ® superflow (IBA-Lifesciences, Göttingen, Germany) for Twin-Strep ® constructs in the batch mode.…”

Section: Protein Purificationmentioning

confidence: 99%

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Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells

Kaipa¹,

Krasnoselska

Owens

et al. 2023

Biomolecules

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Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep® tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.

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Section: Transient Gene Expression In Mammalian Expi293f Cellsmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells

Kaipa¹,

Krasnoselska

Owens

et al. 2023

Biomolecules

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“…This may not be the optimal harvest point for all MP constructs but is a good time-point at which most MPs will have expressed. Specific optimization of expression conditions is something that should be investigated later if higher protein yields are required [12].…”

Section: Notesmentioning

confidence: 99%

“…1). Twelve to 24 constructs per MP target are cloned into the pOPIN vector system before a microscale (3 mL) expression in Expi293F cells [11,12]. Expressed MP constructs are solubilized with β-dodecyl-maltoside (DDM) and Fos-Choline-12 (FC-12) before immobilization by metal affinity using 96-well filter plates as the chromatography column.…”

mentioning

confidence: 99%

High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells

Gamage

Cheruvara

Harrison

et al. 2023

Methods in Molecular Biology

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High-quality protein samples are an essential requirement of any structural biology experiment. However, producing high-quality protein samples, especially for membrane proteins, is iterative and time-consuming. Membrane protein structural biology remains challenging due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Overcoming the twin problems of compositional and conformational instability requires an understanding of protein size, thermostability, and sample heterogeneity, while a parallelized approach enables multiple conditions to be analyzed simultaneously. We present a method that couples the high-throughput cloning of membrane protein constructs with the transient expression of membrane proteins in human embryonic kidney (HEK) cells and rapid identification of the most suitable conditions for subsequent structural biology applications. This rapid screening method is used routinely in the Membrane Protein Laboratory at Diamond Light Source to identify the most successful protein constructs and conditions while excluding those that will not work. The 96-well format is easily adaptable to enable the screening of constructs, pH, salts, encapsulation agents, and other additives such as lipids.

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“…On the other hand, GFP and FPs in general are known to be tightly folded and more resistant to denaturation than most proteins (Saeed and Ashraf, 2009;Ward, 2005). For example, a few studies exploited the ability of GFP to resist the adverse conditions of SDS-PAGE to run protein overexpression screens in Escherichia coli or other cell types (Bird et al, 2015;Bomholt et al, 2013;Geertsma et al, 2008;Krasnoselska et al, 2021;Madani et al, 2021;Muller-Lucks et al, 2012;Newstead et al, 2007), or on recombinantly expressed or purified proteins in vitro (Campbell et al, 2002;Donate-Macian et al, 2019;Nakatani et al, 2019;Weinberger Ii and Lennon, 2021).…”

Section: Introductionmentioning

confidence: 99%

Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

Sanial,

Miled,

Alves

et al. 2024

Preprint

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The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green and red FPs can be maintained in a native, fluorescent form during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent to immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

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Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies

Cited by 8 publications

References 39 publications

Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells

Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells

High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells

Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

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