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Membrane Protein Expression and Purification |
Producing membrane proteins, such as G protein-coupled receptors (GPCRs), can be difficult as it can be hard to preserve the correct conformation of the protein in isolation from its native environment. We provide optimized and trusted membrane protein expression and purification workflow solutions and services backed by a team of experts to express functional membrane proteins in an efficient and reliable manner.
- Optimized workflow—fast and simple protocol to enable to enable native folding and maximum protein yield
- Scalability—scale up and scale down easily
- Versatility—multiple systems to suit your membrane protein research needs
What are membrane proteins?
Membrane proteins (including GPCRs and ion channels) make up approximately a third of all human proteins and are critical to many cellular processes such as ion transport, signal transduction, and catalysis of chemical reactions. Because of their many cellular functions, the study of membrane proteins plays an important role in drug discovery. Approximately half of all pharmaceuticals achieve their therapeutic effects by interacting with membrane proteins.
Four-step workflow to optimize membrane protein expression and membrane protein purification
Membrane proteins are a notoriously difficult class of protein to express, purify, and characterize. Whether you are new to membrane protein expression or an experienced researcher wanting to optimize your protocol, consider this four-step workflow to help maximize the success of your membrane protein expression and purification experiments.
Do you need guidance on a new project, troubleshooting expertise, or more information on a product?
Share your protein expression application needs with us, and one of our protein expression specialists will contact you to discuss the solutions we can provide.
Gene synthesis and cloning—the first step in membrane protein expression
To maximize production yield, genes of interest or DNA starting material are typically subjected to codon optimization to enhance transcription in the expression host.
There are several ways to obtain the DNA starting material for your expression experiments: the gene of interest may be isolated and placed in the vector using standard molecular biology techniques, such as PCR, or the gene may be synthetically produced and cloned into the appropriate vector.
Gene synthesis and cloning tips for membrane protein expression
- Create the best DNA sequence—use the GeneArt codon optimization algorithm to generate the best DNA sequence for your recombinant protein. By using our GeneOptimizer algorithm, you not only adapt the gene to the codon usage of your host system, but you also remove elements that potentially inhibit expression (e.g., killer motifs, splice sites, and RNA secondary structures). Overall, the GeneOptimizer algorithm takes more than 50 parameters into account to determine the optimal gene sequence for more reliable and higher-level protein expression without altering the protein sequence.
- E. coli transformation—Choose the right competent cells for your cloning applications and workflow as they are a critical component to the success of your experiments. There are many aspects to consider when selecting your competent E. coli strain.
- Plasmid preparation and purification—Use endotoxin-free plasmid purification kits such as the PureLink Hi Pure Kits to purify high quality plasmid DNA. Be sure to confirm DNA quality by gel electrophoresis and spectrophotometer (A260/A280) and (A260/A230) readings. Verify the DNA sequence of the final product prior to transfection.
Recommended products and services
Construct creation services
Selecting the right vector is very important in your experimental design. We offer multiple options for vector delivery, once the gene has been synthesized, to supply the maximum flexibility and speed of delivery.
Construct creation kit
Gibson Assembly Cloning is a refined, robust, scarless and seamless cloning methodology that can be utilized in the assembly of multiple DNA fragments, regardless of length or end compatibility in a highly efficient manner.
Vector(s)
pcDNA3.4 and pcDNA5/TO are vectors used within EXPi293 systems and are recommended for optimized membrane protein expression in these systems.
pcDNA3.4 vector for Expi293 parental
pcDNA5/TO vector for Expi293 inducible platform (tetracycline inducible protein expression)
Competent cells
One Shot TOP10 Chemically Competent E. coli delivers advanced genomic DNA cloning capabilities and maximizes cloning efficiency in a single tube format.
Plasmid purification
The PureLink Fast Low-Endotoxin Plasmid Purification kit’s protocol is designed to be simple and fast, providing up to 0.4 mg (midi prep) and 1.5 mg (maxi prep) of high-quality low-endotoxin plasmid DNA suitable for standard transfection of robust cell lines and applications such as PCR, in vitro transcription, cloning, and sequencing.
Automated plasmid purification
The KingFisher system technology provides an efficient workflow solution as one of the most versatile benchtop automated systems in the lab for consistent extraction and purification of DNA, RNA, protein, and cells.
Webinar: Membrane proteins: From gene to cryo-EM structure with Thermo Fisher GeneArt and Salipro Biotech
In this webinar, we demonstrate that the combined expertise of Thermo Fisher Scientific and Salipro Biotech can be utilized for the Gene-to-Structure generation of membrane protein drug targets. Starting with the gene, the GeneArt platform comprises a gene-to-protein service that only requires a protein sequence and covers every step from gene synthesis to protein purification, involving Thermo Fisher’s proprietary mammalian and insect expression systems: Expi293, ExpiCHO, or ExpiSF9.
Optimal and reliable membrane protein expression
Mammalian cells, such as HEK-293 cells and CHO cells, are widely used as hosts to express recombinant proteins. HEK-293 cells in particular are an attractive system for membrane protein expression as they have post-translational modification machineries required for the proper folding and/or optimal biological activity of target proteins.
Recommended products and services
The Expi293 Expression System is precisely designed with suspension 293 cells, optimized medium and transfection reagents to enable native folding and maximum protein yield, allowing your studies to get the biological relevance it deserves.
Experience the Expi293 suite of structural biology products, precisely designed to enable uniform glycosylation, tight control of expression, and protein labeling in the first-ever HEK293 suspension system. Achieve rapid, high-yield protein production with three specialized Expi293F cell lines (GnTI-, Inducible, and Inducible GnTI-) and a Met (-) protein labeling kit.
Expedite your membrane protein expression by partnering with us. We use combination of gene optimization, synthetic DNA expertise and advanced Expi293 Expression Systems to get higher protein yield so you can confidently accelerate your research.
Read about strategies for optimized protein expression in these two posters
Tips for optimal membrane protein expression
Cell culture
- Maintain correct culture volume: flask size, proper shaking speed, and incubator settings for best results.
- Provide fresh media to the cells on Day 1 and Day 0 of the transfection workflow.
- Be mindful of how different amounts of enhancer addition, from what is expected, will yield inferior results ( Expi293 poster Figure 11).
- Consider how Yeliseev (in a collaboration with Thermo Fisher Scientific) shifted the temperature of his incubator from 37°C to 32°C on the day after transfection (Day 1) to improve the expression of membrane proteins. It slows the cells down and allows them to focus on expressing protein, rather than growing [1].
Transfection
- Allow 10–20 minutes for DNA/Expifectamine complex formation.
- Follow the Expi293 user guide and Expi293 E-Learn for best transfection practices in Expi293.
- Consult the Expi293 structural biology and inducible expression user guide for best transfection practices in Expi293.
- Use only high-quality plasmid DNA preps; see plasmid purification section and determine best DNA concentration for optimal transfection efficiency ( Expi293 poster Figure 10).
Harvest
- Run a time course experiment to identify the proper harvest time for your protein of interest based on yield and activity.
Selecting the right membrane protein purification strategy to optimize yield and activity
After protein expression, the next step is to isolate and purify the membrane protein. Protein yield and activity can be maximized at this stage by selecting the right lysis reagents, cleanup procedures, and appropriate purification resin.
Harvest membrane proteins using a strategy ideal for the sample type, protein location, required yield and downstream applications.
Detergents for protein solubilizations
Thermo Scientific LMNG/CHS Solution (10:1) is a ready-to-use formulation for the solubilization of important membrane proteins while preserving structural integrity and activity.
Thermo Scientific Lauryl Maltose Neopentyl Glycol (LMNG) is amphiphilic detergent best suited to boost solubilization of integral membrane proteins while preserving structural integrity and activity.
Thermo Scientific DDM/CHS Solution (10:1) is a ready-to-use solution for solubilization of membrane proteins while preserving structural integrity and activity.
Learn more: Detergents for protein solubilization
Membrane protein extraction and isolation
Thermo Scientific GPCR Extraction and Stabilization Reagent allows efficient extraction and stabilization of G-coupled protein receptors (GPCRs) and other membrane-associated proteins from cultured mammalian cells or tissue while maintaining longer-term receptor integrity.
The Thermo Scientific Pierce Cell Surface Protein Biotinylation and Isolation Kit allows the selective biotinylation, solubilization, and enrichment of plasma membrane proteins.
The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit produces minimal cross-contamination, less than 10% typically, of cytosolic protein into the membrane protein fraction in extraction and isolation.
Learn more: Membrane protein extraction and isolation
Webinar: Strategies for isolation of plasma membrane proteins
This webinar will focus on robust and optimized techniques for extraction, isolation, and enrichment of cell surface proteins, including stable and functional G protein-coupled receptors (GPCRs).
Purification and clean-up
IMAC purification
These EDTA-compatible, high-capacity Ni-IMAC magnetic beads and resins are made to purify overexpressed His-tagged proteins from mammalian or insect cell cultures. Purification can be performed using up to 20 mM EDTA and 20 mM DTT with no loss in performance.
Affinity purification
Thermo Scientific Pierce Anti-DYKDDDDK Affinity Resin is a fast, convenient method for purification and immunoprecipitation (IP) of DYKDDDDK-tagged proteins expressed in in vitro protein expression systems, bacteria, yeast, and mammalian cells.
Clean-up
Dialysis, diafiltration, and gel filtration (desalting) are the most broadly applicable methods for protein clean-up. These techniques are rooted in well-recognized principles of size exclusion and have been utilized in laboratory research for many decades. Advanced quality of materials and designs used to make dialysis, diafiltration, and desalting devices have kept pace with the changes in scale, refinement, and convenience that modern research experiments require.
Overview of dialysis, desalting, buffer exchange, and protein concentration
Quantify, detect, and characterize purified membrane proteins and assess their potency
Protein characterization is an integral part of any laboratory workflow, involving protein extraction and purification. Proteins in cell lysates or purified proteins are identified and quantified to verify yield or normalize multiple samples for side-by-side comparison. Popular protein quantitation and characterization methods in a recombinant protein expression workflow include:
- Protein quantitation assays
- Gel electrophoresis
- Western blotting
- ELISAs
Assays
The Pierce Rapid Gold BCA Protein Assay Kit is a quick, two-component, high-precision, detergent-compatible assay enhanced to establish total protein concentration.
Cryo-EM
The Thermo Scientific VitroEase Buffer Screening Kit offers pre-formulated buffers and detergents and a complete screening strategy to pinpoint optimal protein conditions for single particle cryo-EM sample analysis.
Native mass spectrometry
Intact protein analysis solutions provide minimal protein complexity and resolve the issue of identity. In addition, they offer ultra high resolution of multiple proteins.
The Pierce Detergent Compatible Bradford Assay Kit is a fast and ready-to-use modification of the well-recognized Bradford Coomassie dye-binding, colorimetric method for total protein quantitation.
Thermo Scientific VitroEase Methylamine Tungstate Negative Stain features an easy, ready-to-use negative stain for electron microscopy (EM) analysis.
This page discusses top-down mass spectrometry analysis and its main advantages, including detection of degradation products and sequence variants.
These products include total protein assay kits and reagents, utilized to accurately establish and measure total protein concentration following cell lysis, labeling or purification.
Thermo Scientific VitroEase Methylamine Vanadate Negative Stain features an easy, ready-to-use negative stain for electron microscopy (EM) analysis.
Learn more: Integrative Structural Biology
Gene synthesis and cloning—the first step in membrane protein expression
To maximize production yield, genes of interest or DNA starting material are typically subjected to codon optimization to enhance transcription in the expression host.
There are several ways to obtain the DNA starting material for your expression experiments: the gene of interest may be isolated and placed in the vector using standard molecular biology techniques, such as PCR, or the gene may be synthetically produced and cloned into the appropriate vector.
Gene synthesis and cloning tips for membrane protein expression
- Create the best DNA sequence—use the GeneArt codon optimization algorithm to generate the best DNA sequence for your recombinant protein. By using our GeneOptimizer algorithm, you not only adapt the gene to the codon usage of your host system, but you also remove elements that potentially inhibit expression (e.g., killer motifs, splice sites, and RNA secondary structures). Overall, the GeneOptimizer algorithm takes more than 50 parameters into account to determine the optimal gene sequence for more reliable and higher-level protein expression without altering the protein sequence.
- E. coli transformation—Choose the right competent cells for your cloning applications and workflow as they are a critical component to the success of your experiments. There are many aspects to consider when selecting your competent E. coli strain.
- Plasmid preparation and purification—Use endotoxin-free plasmid purification kits such as the PureLink Hi Pure Kits to purify high quality plasmid DNA. Be sure to confirm DNA quality by gel electrophoresis and spectrophotometer (A260/A280) and (A260/A230) readings. Verify the DNA sequence of the final product prior to transfection.
Recommended products and services
Construct creation services
Selecting the right vector is very important in your experimental design. We offer multiple options for vector delivery, once the gene has been synthesized, to supply the maximum flexibility and speed of delivery.
Construct creation kit
Gibson Assembly Cloning is a refined, robust, scarless and seamless cloning methodology that can be utilized in the assembly of multiple DNA fragments, regardless of length or end compatibility in a highly efficient manner.
Vector(s)
pcDNA3.4 and pcDNA5/TO are vectors used within EXPi293 systems and are recommended for optimized membrane protein expression in these systems.
pcDNA3.4 vector for Expi293 parental
pcDNA5/TO vector for Expi293 inducible platform (tetracycline inducible protein expression)
Competent cells
One Shot TOP10 Chemically Competent E. coli delivers advanced genomic DNA cloning capabilities and maximizes cloning efficiency in a single tube format.
Plasmid purification
The PureLink Fast Low-Endotoxin Plasmid Purification kit’s protocol is designed to be simple and fast, providing up to 0.4 mg (midi prep) and 1.5 mg (maxi prep) of high-quality low-endotoxin plasmid DNA suitable for standard transfection of robust cell lines and applications such as PCR, in vitro transcription, cloning, and sequencing.
Automated plasmid purification
The KingFisher system technology provides an efficient workflow solution as one of the most versatile benchtop automated systems in the lab for consistent extraction and purification of DNA, RNA, protein, and cells.
Webinar: Membrane proteins: From gene to cryo-EM structure with Thermo Fisher GeneArt and Salipro Biotech
In this webinar, we demonstrate that the combined expertise of Thermo Fisher Scientific and Salipro Biotech can be utilized for the Gene-to-Structure generation of membrane protein drug targets. Starting with the gene, the GeneArt platform comprises a gene-to-protein service that only requires a protein sequence and covers every step from gene synthesis to protein purification, involving Thermo Fisher’s proprietary mammalian and insect expression systems: Expi293, ExpiCHO, or ExpiSF9.
Optimal and reliable membrane protein expression
Mammalian cells, such as HEK-293 cells and CHO cells, are widely used as hosts to express recombinant proteins. HEK-293 cells in particular are an attractive system for membrane protein expression as they have post-translational modification machineries required for the proper folding and/or optimal biological activity of target proteins.
Recommended products and services
The Expi293 Expression System is precisely designed with suspension 293 cells, optimized medium and transfection reagents to enable native folding and maximum protein yield, allowing your studies to get the biological relevance it deserves.
Experience the Expi293 suite of structural biology products, precisely designed to enable uniform glycosylation, tight control of expression, and protein labeling in the first-ever HEK293 suspension system. Achieve rapid, high-yield protein production with three specialized Expi293F cell lines (GnTI-, Inducible, and Inducible GnTI-) and a Met (-) protein labeling kit.
Expedite your membrane protein expression by partnering with us. We use combination of gene optimization, synthetic DNA expertise and advanced Expi293 Expression Systems to get higher protein yield so you can confidently accelerate your research.
Read about strategies for optimized protein expression in these two posters
Tips for optimal membrane protein expression
Cell culture
- Maintain correct culture volume: flask size, proper shaking speed, and incubator settings for best results.
- Provide fresh media to the cells on Day 1 and Day 0 of the transfection workflow.
- Be mindful of how different amounts of enhancer addition, from what is expected, will yield inferior results ( Expi293 poster Figure 11).
- Consider how Yeliseev (in a collaboration with Thermo Fisher Scientific) shifted the temperature of his incubator from 37°C to 32°C on the day after transfection (Day 1) to improve the expression of membrane proteins. It slows the cells down and allows them to focus on expressing protein, rather than growing [1].
Transfection
- Allow 10–20 minutes for DNA/Expifectamine complex formation.
- Follow the Expi293 user guide and Expi293 E-Learn for best transfection practices in Expi293.
- Consult the Expi293 structural biology and inducible expression user guide for best transfection practices in Expi293.
- Use only high-quality plasmid DNA preps; see plasmid purification section and determine best DNA concentration for optimal transfection efficiency ( Expi293 poster Figure 10).
Harvest
- Run a time course experiment to identify the proper harvest time for your protein of interest based on yield and activity.
Selecting the right membrane protein purification strategy to optimize yield and activity
After protein expression, the next step is to isolate and purify the membrane protein. Protein yield and activity can be maximized at this stage by selecting the right lysis reagents, cleanup procedures, and appropriate purification resin.
Harvest membrane proteins using a strategy ideal for the sample type, protein location, required yield and downstream applications.
Detergents for protein solubilizations
Thermo Scientific LMNG/CHS Solution (10:1) is a ready-to-use formulation for the solubilization of important membrane proteins while preserving structural integrity and activity.
Thermo Scientific Lauryl Maltose Neopentyl Glycol (LMNG) is amphiphilic detergent best suited to boost solubilization of integral membrane proteins while preserving structural integrity and activity.
Thermo Scientific DDM/CHS Solution (10:1) is a ready-to-use solution for solubilization of membrane proteins while preserving structural integrity and activity.
Learn more: Detergents for protein solubilization
Membrane protein extraction and isolation
Thermo Scientific GPCR Extraction and Stabilization Reagent allows efficient extraction and stabilization of G-coupled protein receptors (GPCRs) and other membrane-associated proteins from cultured mammalian cells or tissue while maintaining longer-term receptor integrity.
The Thermo Scientific Pierce Cell Surface Protein Biotinylation and Isolation Kit allows the selective biotinylation, solubilization, and enrichment of plasma membrane proteins.
The Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit produces minimal cross-contamination, less than 10% typically, of cytosolic protein into the membrane protein fraction in extraction and isolation.
Learn more: Membrane protein extraction and isolation
Webinar: Strategies for isolation of plasma membrane proteins
This webinar will focus on robust and optimized techniques for extraction, isolation, and enrichment of cell surface proteins, including stable and functional G protein-coupled receptors (GPCRs).
Purification and clean-up
IMAC purification
These EDTA-compatible, high-capacity Ni-IMAC magnetic beads and resins are made to purify overexpressed His-tagged proteins from mammalian or insect cell cultures. Purification can be performed using up to 20 mM EDTA and 20 mM DTT with no loss in performance.
Affinity purification
Thermo Scientific Pierce Anti-DYKDDDDK Affinity Resin is a fast, convenient method for purification and immunoprecipitation (IP) of DYKDDDDK-tagged proteins expressed in in vitro protein expression systems, bacteria, yeast, and mammalian cells.
Clean-up
Dialysis, diafiltration, and gel filtration (desalting) are the most broadly applicable methods for protein clean-up. These techniques are rooted in well-recognized principles of size exclusion and have been utilized in laboratory research for many decades. Advanced quality of materials and designs used to make dialysis, diafiltration, and desalting devices have kept pace with the changes in scale, refinement, and convenience that modern research experiments require.
Overview of dialysis, desalting, buffer exchange, and protein concentration
Quantify, detect, and characterize purified membrane proteins and assess their potency
Protein characterization is an integral part of any laboratory workflow, involving protein extraction and purification. Proteins in cell lysates or purified proteins are identified and quantified to verify yield or normalize multiple samples for side-by-side comparison. Popular protein quantitation and characterization methods in a recombinant protein expression workflow include:
- Protein quantitation assays
- Gel electrophoresis
- Western blotting
- ELISAs
Assays
The Pierce Rapid Gold BCA Protein Assay Kit is a quick, two-component, high-precision, detergent-compatible assay enhanced to establish total protein concentration.
Cryo-EM
The Thermo Scientific VitroEase Buffer Screening Kit offers pre-formulated buffers and detergents and a complete screening strategy to pinpoint optimal protein conditions for single particle cryo-EM sample analysis.
Native mass spectrometry
Intact protein analysis solutions provide minimal protein complexity and resolve the issue of identity. In addition, they offer ultra high resolution of multiple proteins.
The Pierce Detergent Compatible Bradford Assay Kit is a fast and ready-to-use modification of the well-recognized Bradford Coomassie dye-binding, colorimetric method for total protein quantitation.
Thermo Scientific VitroEase Methylamine Tungstate Negative Stain features an easy, ready-to-use negative stain for electron microscopy (EM) analysis.
This page discusses top-down mass spectrometry analysis and its main advantages, including detection of degradation products and sequence variants.
These products include total protein assay kits and reagents, utilized to accurately establish and measure total protein concentration following cell lysis, labeling or purification.
Thermo Scientific VitroEase Methylamine Vanadate Negative Stain features an easy, ready-to-use negative stain for electron microscopy (EM) analysis.
Learn more: Integrative Structural Biology
References
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