Name: expression and purification of mammalian bestrophin ion channels
Uploaded: 2018-08-02T14:00:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

This project was funded by NIH grants EY025290, GM127652, and University of Rochester start-up funding.

1. Producing BacMam Expression Baculoviruses
<ol>
	<li>Insert the coding sequence of a desired mammalian bestrophin protein into the pEG BacMam vector18 with a Tobacco Etch Virus (TEV) protease recognition sequence, followed by a GFP-10x His-tag at the C-terminus of the protein.</li>
	<li>Transiently transfect the expression plasmid into adhesive HEK293 cells19,20,21,22,23. Check the expression of the GFP-fusion protein under a fluorescent microscope with 10X or 20X magnification, a 488 nm excitation source, and a 510 nm emission filter (Figure 1A). 
	Note: Polymer-based transfection is routinely performed with 1 &#956;g of plasmid DNA for a 35&#160;mm dish of HEK293 cells.</li>
	<li>Produce the baculoviruses in Sf9 insect cells as previously described16,17. 
	Note: The passage 3 (P3) virus, which will be used to infect the HEK293-F cells for protein production, can be stored at 4 &#176;C for 1 month.</li>
	<li>Determine the titer (infectious units) of the P3 virus by the plaque formation assay16,17. 
	Note: As the protein of interest is tagged with GFP, a faster method for checking infectious units is to infect Sf9 cells with serial dilutions of the virus and count the number of fluorescent cells after 48 h.</li>
</ol>
2. Protein Expression
<ol>
	<li>Maintain the HEK293-F cells in a suspension culture with the HEK293-F expression medium in a 37 &#176;C humidified incubator with 8% CO2. Use disposable culture flasks that are 2-2.5 times bigger than the culture volume and rotate the culture on an orbital platform at 135 rpm. 
	Note: It is recommended to infect the cells when they are between passages 5 and 30 and to perform these actions under a cell culture hood.</li>
	<li>24 h before viral infection, add 15 &#956;L of Trypan blue to a 15 &#956;L aliquot of cell culture, and check the cell density and viability using a hemocytometer under a microscope. Dilute the cells to 0.6 x 106 cells/mL in 2 x 2 L flasks each, with 500 mL of medium pre-warmed at 37 &#176;C. 
	Note: Dead cells are stained blue by Trypan blue. The desired cell viability is &#62;95%.</li>
	<li>On the day of infection, check the cell density and viability (step 2.2). 
	Note: A high cell viability of &#62;95% is essential for efficient infection and protein expression. The expected cell density is ~1.0 x 106 cells/mL (grown overnight from 2 x 500 mL of the 0.6 x 106 cells/mL cell culture). The expected total number of cells is ~1.0 x 109.</li>
	<li>Add the P3 baculoviruses to the cells at a multiplicity of infection (MOI) of 5. To determine the amount of virus to inoculate, use the following equation: 
	<img alt="Equation" src="/files/ftp_upload/57832/57832eq1.jpg" /></li>
	<li>Shake the cells on the orbital platform at 135 rpm in a humidified 37 &#176;C incubator with 8% CO2 for 24 h.</li>
	<li>Add 10 mM of sodium butyrate to the cell culture and continue incubating for 48 h. 
	Note: For some proteins, reducing the cell culture temperature to 30 &#176;C after sodium butyrate addition may improve expression and stability.</li>
	<li>Under a fluorescence microscope with 10X or 20X magnification, a 488 nm excitation source, and a 510 nm emission filter, check the percentage and brightness of green cells, which directly represent the protein (bestrophin-GFP-10xHis) yield (Figure 1B). 
	Note: The percentage of green cells is calculated by: 100 x (Green cells/Total cells). Good protein expression level is indicated by strong green fluorescence under the microscope.</li>
	<li>Harvest the cells by centrifuging at 1,000 x g in 4 &#176;C for 20 min. Remove the supernatant and re-suspend the cell pellets with phosphate-buffered saline (PBS) to a final volume of ~80 mL. Split the cell suspension into 2 x 50 mL conical tubes and centrifuge at 1,000 x g in 4 &#176;C for 20 min. Store the cell pellets at -80 &#176;C.</li>
</ol>
3. Protein Purification
<ol>
	<li>Incubate the conical tubes containing the cell pellets in a stirring water bath at room temperature for 10-15 min so that they thaw. Re-suspend the cells in 2x (w/v) volume of buffer A (Table 1) (e.g., 10 g of cell pellets in 20 mL of buffer) supplemented with proteinase inhibitors (Aprotinin, Leupeptin, Pepstatin A and phenylmethylsulfonyl fluoride) at 0.1-1.0 mM. Pipet up and down extensively to obtain a homogenous cell suspension.</li>
	<li>Lyse the cells using a high-pressure homogenizer. Run the cell suspension through the homogenizer at 7-10 MPa for a total of 3-4 times to achieve complete homogenization. Keep the cell lysate on ice for 2-3 mins between rounds.</li>
	<li>Add detergent [e.g., 2% w/v sol-grade n-Dodecyl-&#946;-D-Maltopyranoside (DDM)] to the cell lysate. Incubate with agitation for 1 h at 20 &#176;C to extract the membrane proteins. 
	Note: The type of detergent needs to be optimized for each protein target18,24.</li>
	<li>Centrifuge at 150,000 x g in an ultracentrifuge at 4 &#176;C for 1 h. 
	Note: All of the following steps are performed at 4 &#176;C.</li>
	<li>Collect the clear cell lysate and flow it through a 5 mL His Trap Ni2+-NTA affinity column pre-equilibrated with buffer A (Table 1). 
	Note: After centrifugation, the clear cell lysate will be sandwiched between a pellet at the bottom and a cloudy layer on top. Use a 10 mL transfer pipet to carefully collect the clear lysate and then switch to a 1 mL pipet for the last few milliliters of lysate. Avoid the pellet, which can clog the Ni2+ column; however, a small amount of the top layer is fine or can be filtered out with a 1.5 &#956;m filter.</li>
	<li>Wash the column with 25 mL of buffer B and then 40 mL of buffer C (Tables 2 and 3, respectively). 
	Note: This is a good place to take a break, as total purification may take up to 12 h and the protein can remain stable while attached to the column overnight.</li>
	<li>Attach the column to a fast protein liquid chromatography (FPLC) system and elute the protein from the column with 13 mL of buffer D (Table 3) with fractionation. Collect the protein-enriched fractions according to the UV absorbance readout. 
	Note: The FPLC conditions are as follows: a 1.0 mL fraction size, a pre-column pressure alarm set to 0.3 MPa, and a flow rate of 0.5 mL/min.</li>
	<li>Measure the eluted protein product concentration on a microvolume spectrophotometer. Read the absorbance at 280 nm.</li>
	<li>(Optional) To remove the GFP-10xHis tag, add the TEV protease at a 1:1 mass ratio and incubate at 4 &#176;C for 30 min. 
	Note: The tag may or may not affect the function or structure of the purified channel. Calculate the TEV amount from the volume and concentration of the collected protein from steps 3.7-3.8. For instance, if 10 mL of elution product is collected and measured at 0.1 mg/mL, the total protein mass is 10 x 0.1 = 1 mg, and 1 mg of TEV will be used for digestion.</li>
	<li>Concentrate the protein with a 15 mL centrifugal (50 or 100 kDa) filter unit by spinning at 4,000 x g in 4 &#176;C to a final volume of 400-500 &#956;L (for a 2 mL sample-loading loop on FPLC). 
	Note: The centrifuging time for concentration varies depending on the concentration and size of the protein. To avoid over-concentrating, which may cause protein precipitation, spin for 10 min at first, then observe the remaining volume and estimate the time for a subsequent spin (e.g., 2-5 min), and so on, to obtain the final volume. Pipet&#160;between spins to disperse the protein, which is pulled to the bottom of the filter.</li>
	<li>Transfer the concentrated protein to a new 1.5 mL tube and remove any precipitate or bubbles from the concentrated product. Spin at &#62;12,000 x g for 5-10 min at 4 &#176;C and collect the supernatant in a new 1.5 mL tube.</li>
	<li>Load the final product with a 1 mL syringe and a round-tip needle on a FPLC system for size-exclusion chromatography with a size-exclusion column pre-equilibrated with buffer E (Table 5). 
	Note: The FPLC conditions are as follows: a 0.5 mL fraction size, a pre-column pressure alarm set to 2.50 MPa, a flow volume set to 30 mL of buffer E (Table 5), and a flow rate set to 0.5 mL/min.</li>
	<li>Note that well-behaved proteins run as a single peak (Figure 2A) and collect the protein fraction(s) corresponding to that peak (Figure 2A).</li>
	<li>Concentrate the protein with a 4 mL or 0.5 mL centrifugal filter unit (of the same molecular weight cut-off as in step 3.10) and spin at 4,000 x g at 4 &#176;C to a final concentration of 5-10 &#956;g/&#956;L. Use the spectrophotometer to check the final product concentration (step 3.8). 
	Note: Centrifugation time varies, as the final product volume can differ between purification trials. Usually, centrifugation takes 10-20 min. It is recommended to pipet&#160;between spins to disperse the protein, which is pulled to the bottom of the filter.</li>
	<li>Transfer the concentrated protein to a new 1.5 mL tube. Remove any precipitate by centrifuging at &#62;12,000 x g for 5-10 min at 4 &#176;C. Transfer the supernatant to a new 1.5 mL tube. Make 10 &#956;L aliquots for storage at -80 &#176;C and save one 2-5 &#956;L small aliquot to run on a 4-15% gradient SDS-PAGE gel at 9 V/cm (Figure 2B).</li>
	<li>Confirm the identity of the purified protein by mass spectrometry8. 
	Note: In vitro analysis of the function and structure of the purified protein can be performed through planar lipid bilayer and X-ray crystallography, respectively5,8.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes a useful pipeline for expression and purification of mammalian bestrophin ion channels to be used for future in vitro analyses. While the FPLC device is required for size-exclusion chromatography, a syringe pump is sufficient for all steps of affinity chromatography including binding, washing, and eluting. When using a syringe pump to push solutions (in a syringe) through a column, it is essential to underlay the spring side to avoid pushing air bubbles into the column. If the purity of t...

Bestrophins are a family of ion channels conserved through species varying from bacteria to humans1. In humans, the BEST1 gene, located on chromosome 11q12.3, encodes the membrane protein Bestrophin-1 (BEST1) that is predominantly expressed in the basolateral membrane of retinal pigment epithelium (RPE) cells of the eyes2,3,4. Comprised of 585 amino acids, the first ~350 of which are highly conserved among species and contain its transmembrane region, BEST1 acts as a CaCC in humans1,5,6. Furthermore, the BEST1 homologs in chickens and Klebsiella pneumoniae both function as homopentamers7,8, suggesting a high level of conservation throughout evolution.
In humans, over 200 mutations in the BEST1 gene have been clinically linked to a group of retinal degeneration diseases called bestrophinopathies1,9. Five specific bestrophinopathies have been reported, including Best disease, adult-onset vitelliform dystrophy, autosomal dominant vitreoretinochoroidopathy, autosomal recessive bestrophinopathy, and retinitis pigmentosa3,4,10,11,12,13,14. These diseases, which lead to decreased eyesight and even blindness, are currently untreatable. In order to develop therapeutic treatments and potentially personalized medicine, it is critical to understand how these BEST1 disease-causing mutations influence the function and structure of the BEST1 channel15. For these purposes, researchers need to&#160;obtain purified bestrophin (wild type and/or mutant) channels and conduct in vitro analyses5,8.
The first key step is the expression of bestrophin channels from higher species in mammalian cells. As baculovirus transduction of HEK293-F cells (the BacMam system) is a powerful method to heterologously express membrane proteins16,17, this protocol utilizes an optimized BacMam vector (pEG BacMam) for robust expression of the target protein18, which in this case is a mammalian bestrophin homolog. This vector has been used for the expression of various membrane proteins, including G-protein-coupled receptors, nuclear receptors, and other ion channels18. There is also evidence that the produced proteins are suitable for crystallography18. With high levels of expression in HEK293-F cells, the proteins can then be purified using chromatography; specifically, in the case of bestrophins, both affinity and size-exclusion chromatography can be used.
Once this protocol is fine-tuned for a bestrophin channel, the purified protein can then be analyzed for its function and structure through planar lipid bilayer and X-ray crystallography, respectively5,8. Altogether, these techniques provide a powerful pipeline for functional and structural investigations of bestrophins and other ion channels.

<table><tbody><tr><td>HEPES</td><td>Fisher Scientific</td><td>AC327265000&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>AC446212500</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-500</td><td></td></tr><tr><td>Imidazole</td><td>Fisher Scientific</td><td>AC301870010</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>AC197530010&amp;nbsp;</td><td></td></tr><tr><td>TCEP</td><td>Fisher Scientific</td><td>AA4058704&amp;nbsp;</td><td></td></tr><tr><td>Aprotinin</td><td>Fisher Scientific</td><td>AAJ63039MA</td><td></td></tr><tr><td>Leupeptin</td><td>Fisher Scientific</td><td>AAJ61188MB&amp;nbsp;</td><td></td></tr><tr><td>Pepstatin A</td><td>Fisher Scientific</td><td>AAJ20037MB</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Fisher Scientific</td><td>AC215740050</td><td></td></tr><tr><td>DDM sol-grade</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>DDM anagrade</td><td>Anatrace</td><td>D310</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>ThermoFisher</td><td>10902179</td><td></td></tr><tr><td>FreeStyle medium</td><td>ThermoFisher</td><td>12338018</td><td></td></tr><tr><td>NanoDrop spectrophotometer</td><td>ThermoFisher</td><td>ND-2000</td><td></td></tr><tr><td>High pressure homogenizer</td><td>Avestin</td><td>Emulsiflex-C5</td><td></td></tr><tr><td>HisTrap column</td><td>GE</td><td>17-5248-01</td><td></td></tr><tr><td>Superdex-200 column</td><td>GE</td><td>28990944</td><td></td></tr><tr><td>AKTA Pure</td><td>GE</td><td>29018224</td><td></td></tr><tr><td>Ultra-15 centrifugal filter units</td><td>Millipore</td><td>UFC910024</td><td></td></tr><tr><td>Ultra-4 centrifugal filter units</td><td>Millipore</td><td>UFC810024</td><td></td></tr><tr><td>Ultra-0.5 centrifugal filter units</td><td>Millipore</td><td>UFC505024</td><td></td></tr><tr><td>Optima XE-90 Ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658004</td><td></td></tr><tr><td>Mini-PROTEAN precast gel</td><td>Bio-Rad</td><td>4561084</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>PolyJet transfection reagent</td><td>SignaGen</td><td>SL100688</td><td></td></tr><tr><td>pEG BacMam vector</td><td></td><td></td><td>Obtained from the Gouaux lab at Vollum Institute</td></tr></tbody></table>

expression and purification of mammalian bestrophin ion channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The fluorescence intensity in transiently-transfected adhesive HEK293 cells (Figure 1A) is a good indicator for the projected protein expression level in suspension&#160;HEK293-F cells (Figure 1B). If the target protein is not well-expressed or is mis-localized in HEK293 cells after transient transfection, it is recommended to consider modifying the expression construct (e.g., changing the position of the GFP tag or maki...

Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

Expression and Purification of Mammalian Bestrophin Ion Channels

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

expression-and-purification-of-mammalian-bestrophin-ion-channels

Research

JoVE Journal

Biochemistry

8.3K Views.  University of Rochester, School of Medicine and Dentistry. De zuivering van ionenkanalen is vaak uitdagend, maar eenmaal bereikt, kan het potentieel toestaan in vitro onderzoeken van de functies en structuren van de kanalen. Hier beschrijven we de stapsgewijze procedures voor de expressie en de zuivering van eiwitten van zoogdieren bestrophin, een familie van Ca2 +-geactiveerd Cl- -kanalen.

Video: Expression and Purification of Mammalian Bestrophin Ion Channels

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Biology

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Neuroscience

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Expression and Purification of Mammalian Bestrophin Ion Channels

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