The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.
During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.
Size exclusion chromatography (SEC), also known as gel filtration chromatography, is commonly used in protein science1. During SEC, proteins are separated based on their hydrodynamic radius, which is a function of the protein size and shape2. In brief, this separation is achieved by applying the protein samples under flow to a packed bed of porous beads that act as a molecular sieve. The beads used are often cross-linked agarose with a defined range of pore sizes to allow proteins to either enter or be excluded from the pores of the beads3,4,5,6,7. Proteins with smaller hydrodynamic radii spend a greater proportion of time within the pores and, thus, flow through the packed bed at a slower rate, whereas larger proteins spend a greater proportion of time outside the beads (the excluded volume) and move through the packed bed at a faster rate. SEC can be used as a protein purification step when a preparative column is used1. When an analytical column is used, SEC can be used to analyze the protein quality and properties2. For example, protein aggregates that may be present in a sample and indicate poor quality protein tend to be very large, meaning they only travel in the excluded volume and, thus, are eluted from the column at the earliest point; this volume is referred to as the column void or void volume. Furthermore, molecular weight standards can be used to calibrate the column, allowing an estimated molecular weight of the protein of interest to be interpolated from a standard curve.
Typically, the protein absorbance at 280 nm is used to monitor the protein elution from a size exclusion column. This restricts the use of SEC as an analysis tool until the protein of interest is largely free from contaminating proteins, for example, at the last step of protein purification. However, fluorescent SEC (FSEC) utilizes a protein of interest that is fluorescently labeled. Therefore, a fluorescent signal can be used to specifically monitor the elution of the protein of interest in the presence of other proteins or even crude mixtures8,9. Furthermore, as fluorescent signals are highly sensitive, successful analysis can be performed on samples with extremely low protein quantities. The protein of interest is often fluorescently labeled by including a green fluorescent protein (GFP) or enhanced GFP (eGFP) tag in the expression construct. The fluorescent signal can then be monitored by excitation at 395 nm or 488 nm and detecting the fluorescent emission at 509 nm or 507 nm for GFP or eGFP, respectively10.
The benefit of using a fluorescent signal to monitor protein elution from an SEC column makes FSEC a valuable tool for analyzing membrane protein samples when the expression levels are particularly poor in comparison to soluble proteins. Crucially, the quality and properties of membrane proteins can be analyzed directly following solubilization from crude lysates without the requirement to optimize the purification process first11,12. For these reasons, FSEC can be used to rapidly analyze the membrane protein quality while exploring the different factors that may be required to improve the behavior of the membrane protein in solution. For example, it is common to trial numerous constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane13,14. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein can define the best buffer composition for protein purification or for providing stability for downstream uses, such as biophysical assays or structural characterization.
Thus, the overall goal of the FSEC method is to collect an SEC column elution profile for a target membrane protein of interest. Furthermore, as fluorescence is used, this SEC trace is collected at the earliest possible point in the optimization of the constructs and conditions prior to any lengthy purification. The FSEC trace can be used as a comparative tool to judge the likelihood of success of purifying a membrane protein with different buffer conditions or membrane protein constructs. In this way, the collection of FSEC profiles can be used as a quick iterative process to arrive at optimal construct design and buffer composition prior to spending effort generating the quantities of pure protein required for other analysis methods.
1. Detergent and buffer preparation for FSEC
2. Sample preparation for FSEC
3. Size exclusion chromatography (SEC)
4. Fluorescent trace collection and analysis
Figure 1: Schematic representation of the steps required to run an FSEC experiment. (1) Cells that express the fluorescently tagged protein of interest are solubilized. (2) The crude solubilization is clarified first with a low-speed spin, followed by (3) a high-speed spin. (4) The clarified sample supernatant is loaded and run on an appropriate SEC column, and (5) the fractions are collected. (6) Samples of the fractions are transferred to a 96-well plate, and a GFP-fluorescent signal is detected using a plate reader to (7) plot the FSEC trace. Please click here to view a larger version of this figure.
First, the dynamic range and lower limits of eGFP detection for the plate reader used in this study were investigated. A purified eGFP standard of known concentration was diluted in a 50 µL final volume to 50 ng·µL−1, 25 ng·µL−1, 12.5 ng·µL−1, 6.25 ng·µL−1, 3.125 ng·µL−1, 1.5625 ng·µL−1, 0.78125 ng·µL−1, and 0.390625 ng·µL−1, and the fluorescence was read using an excitation of 488 nm and an emission of 507 nm (Figure 2). This experiment indicated that the plate reader had a lower detection limit of 30 ng of eGFP-labeled protein per well and a dynamic range of up to 500 ng of eGFP-labeled protein per well before signal saturation. Using the value for the lower limit and assuming that the protein elution is confined to 0.33 of the column volume, as little as 1.28 µg of eGFP labeled protein of interest is required for SEC column loading in order for a detectable FSEC signal to be observed.
Figure 2: eGFP standard curve. Scatter graph of the fluorescent signal for purified eGFP standard diluted to 50 ng·µL−1, 25 ng·µL−1, 12.5 ng·µL−1, 6.25 ng·µL−1, 3.125 ng·µL−1, 1.5625 ng·µL−1, 0.78125, and 0.390625 ng·µL−1. (A) All dilutions are displayed, including those with the saturated signal. (B) A zoomed-in scatter graph including only the standards that fall into the dynamic range of the plate reader. Please click here to view a larger version of this figure.
Secondly, the 24 mL column used for this study was calibrated with molecular weight standards. Using the same buffer and running conditions as used for the FSEC analysis, the molecular weight standards blue dextran (>2,000 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) were individually injected and run through the column, and elution traces were collected at 280 nm absorption. The elution volumes recorded were 8.9 mL, 12.4 mL, 15.2 mL, 16.9 mL, and 18 mL, respectively. When these elution volumes were converted to Kav (Equation 1) and plotted against log molecular weights, a standard curve could be fit. This allowed the molecular weight of the GPCRs tested in this study to be estimated by interpolation of the standard curve (Figure 3). For example, the FSEC trace of the GPCR serotonin receptor 2A (5HT2AR) after solubilization in the detergent DDM indicated an elution volume of 13.4 mL. This 5HT2AR elution volume falls between those elution volumes recorded for ferritin and aldolase and provides an estimated molecular weight of approximately 300 kDa. The 5HT2AR construct used in this study is approximately 50 kDa (including the eGFP tag), meaning that if 5HT2AR is assumed to be monomeric, 250 kDa of molecular weight could be attributed to the DDM detergent/lipid micelle. The equation for the conversion of elution volumes is as follows (Equation 1):
(Equation 1)
where Ve is the elution volume, V0 is the column void volume, and Vc is the total column volume.
Figure 3: Calibration curve of the SEC column using molecular weight standards. (A) A representative FSEC trace of 5HT2AR solubilized in DDM, with the relative elution positions of the molecular weight standards blue dextran (Void), ferritin, aldolase, conalbumin, and ovalbumin marked. Ferritin, aldolase, conalbumin, and ovalbumin are colored in green, purple, red, and cyan, respectively. (B) The molecular weight calibration curve using the elution positions of the standards after conversion to Kav (Equation 1) plotted against the log molecular weight (Mr). The Mr of 5HT2AR in DDM was interpolated from the curve using Kav and is displayed on the curve (blue square). Please click here to view a larger version of this figure.
FSEC was then used to assess the quality and properties of the GPCR sphingosine-1-phosphate receptor (S1PR1)15. Insect cells expressing GFP-tagged human S1PR1 were processed for FSEC as described in the protocol (Figure 1).
Firstly, the optimal membrane extraction conditions were explored by testing the detergents DDM and LMNG against detergent-free extraction with SMA (Figure 4A). The monodispersity index was used to assess the quality of the protein sample and the ratio between the protein in the void (~8 mL column retention) compared with the monodispersed sample (14-15 mL column retention). The sample solubilized in LMNG displayed a superior FSEC profile with a better monomer peak shape and a lower protein aggregate peak, indicating that solubilization and purification in LMNG were the most stabilizing conditions for this membrane protein. In contrast, the sample solubilized in DDM had a relatively poorer FSEC profile, with a larger aggregate peak and a broader monomeric peak, indicating polydispersity in the sample.
Secondly, the effect of ligand addition on the FSEC profile was investigated by adding sphingosine-1-phosphate (S1P) to the sample during solubilization. In this instance, DDM was used as the solubilization reagent, and the FSEC traces of S1PR1 in the presence and absence of S1P were compared (Figure 4B). The sample solubilized in the presence of S1P showed a superior FSEC trace with reduced aggregation. This indicated that purification in the presence of a ligand was advantageous for improving the protein sample quality by stabilizing the receptor in solution but was also advantageous also as a surrogate marker of protein activity, as the results suggested the protein was correctly folded and competent for ligand binding.
Figure 4: FSEC using a crude extract of S1PR1 indicating optimal membrane extraction conditions and ligand binding. (A) Comparison of the FSEC traces of S1PR1 solubilized in styrene-maleic acid co-polymer (SMA; blue), lauryl maltose neopentyl glycol (LMNG; red), or dodecyl maltoside (DDM; black). (B) Comparison of the FSEC traces of S1PR1 solubilized in DDM in the presence (red) or absence (black) of the agonist sphingosine-1-phosphate (S1P). Please click here to view a larger version of this figure.
FSEC was also used to investigate the long-term stability of 5HT2AR under different conditions. GFP-tagged human 5HT2AR was solubilized from insect cell membranes in either detergent (DDM) or SMA polymer and analyzed by FSEC at several time points after storage at 4 °C or room temperature (Figure 5). Following several days of incubation, 5HT2AR in DDM displayed a significant drop in the monomeric peak height at either temperature, and significant increases in the aggregate peak were observed. In contrast, 5HT2AR in SMA lipid particle (SMALP) did not show a significant drop in the monomeric peak height over the course of the experiment, indicating that the protein in SMALP remained stable for longer, even at unfavorable temperatures. This may be important when considering protein preparations for downstream biophysical applications such as surface plasmon resonance (SPR) experiments for which there is a requirement for the sample to be stable and active over a long time period for the successful completion of the binding experiments.
Figure 5: Effect of time and temperature on the quality of 5HT2AR extracted from the membrane using either DDM or SMALP, as analyzed by FSEC. (A) Representative FSEC traces of 5HT2AR solubilized in DDM and stored at room temperature for 1 day (grey), 2 days (green), or 5 days (blue). (B) Histograms of the normalized monomeric peak height for DDM samples stored at 4 °C (blue) or RT (green) compared to SMALP samples stored at 4 °C (grey) or RT (black). Error bars are representative of the SEM. Please click here to view a larger version of this figure.
The generic systematic approaches for condition screening with FSEC that are presented here allow the fast optimization of solubilization and purification parameters for the production of membrane proteins. This means that stable and functionally active membrane proteins can be rapidly produced for biophysical and structural studies. Furthermore, FSEC can be run using laboratory equipment that is likely already in place in membrane protein labs, and thus, there is no requirement for the purchase of a specialist instrument for running the assays.
Critical steps
The time taken between the point of solubilization from cells in detergent to the point at which the sample is passed down the SEC column (steps 2.1.5-3.2.5) are time critical, and there must be no pauses between these steps. All the steps should be conducted at 4 ˚C or on ice, and the time taken to perform these steps needs to be kept to the minimum possible. These time and temperature constraints are necessary in order to record the FSEC profile for the membrane protein before any potential unfolding or degradation. After the membrane protein has been solubilized, there is a greater risk of unfolding, aggregation, and degradation, even at 4 ˚C. Ideally, any samples for which the FSEC traces are to be compared should pass down the SEC column in the same length of time after the solubilization step. In practice, this is difficult, particularly if the samples are passed sequentially down a single column, but it is possible to collect up to five SEC traces within 3 h of one another, and in this time frame, there should not be significant degradation.
Troubleshooting
If, on performing the FSEC experiment, there is low or no fluorescent signal, it is possible the membrane protein of interest has not expressed the chosen cell line, has very low expression of the chosen cell line, or has not been solubilized in the chosen detergent. If the samples were diluted before collecting the fluorescence signal and recording the FSEC trace, a simple first step would be to try a lower dilution or no dilution of the SEC fractions. If this still does not yield an interpretable FSEC trace, the expression and solubilization of the protein should be checked.
The analysis of protein expression can be achieved by checking the fluorescence of the sample after step 2.2.2. If there is a very low or no fluorescent signal from this sample (e.g., a signal very close to the background), there is likely an issue with the protein expression. Steps can be taken to improve the expression levels of the membrane protein, such as switching to an alternate cell line or adjusting the growth conditions, the induction of expression, and the time between the induction/infection/transfection and the harvest. However, particularly poor protein expression can indicate an unstable membrane protein and, thus, a poor construct choice.
If the expression has been checked and there is a clear fluorescent signal above the background prior to FSEC, the solubilization efficiency can be checked by measuring the remaining fluorescent signal of the sample after step 2.4.3 (soluble membrane protein) in comparison to the sample after step 2.2.2 (total protein). It is common for the solubilization efficiency to be 20%-30% and still allow for the successful analysis and purification of the membrane protein. However, if the solubilization efficiency is less than 20%, a different detergent for solubilization or different solubilization conditions may be required. If attempts to improve the solubilization are not successful, this can indicate a particularly unstable membrane protein and, thus, a poor construct choice.
If a very late eluting peak is observed in the FSEC trace (e.g., 18-24 mL), this indicates that the fluorescent protein has a protein molecular weight that is much lower than expected. This can be caused by the membrane protein of interest being degraded, resulting in "free" GFP. One should check if the protein is intact before and after solubilization using in-gel GFP fluorescence. If the protein of interest does appear to be degrading or being proteolyzed, the amount of protease inhibitor can be increased twofold to fourfold. However, a high sensitivity to proteases or degraded protein even before solubilization can indicate a particularly unstable protein and, thus, a poor construct choice.
Modifications and further applications of FSEC
Commonly, the fluorescent tag that is used in FSEC is GFP or eGFP, as described in this protocol. However, many different fluorescent protein tags are available. The choice of the fluorescent tag to be used depends on having a plate reader that can achieve the correct excitation and emission parameters to record the fluorescent signal for the selected fluorescent tag and having a fluorophore with little to no change in quantum yield in different environmental conditions. Furthermore, FSEC is not restricted to fluorescent proteins but can also work equally well with a protein that has been labeled with a fluorescent dye. For example, an NTA dye could be used, which would favorably bind to histidine-tagged membrane protein constructs. Furthermore, either a fluorescently labeled antibody chemically labeled with a fluorescent dye and specific for binding the membrane protein of interest or a purification tag included in the membrane protein construct could indirectly label a target for FSEC.
When performing detergent screening using FSEC, a choice can be made regarding whether the buffer used to run the SEC column should contain the matching detergent that the protein has been solubilized in or whether a standard detergent should be used across all the runs. A more accurate representation of the behavior of the protein will be obtained if the whole experiment is performed with the matching detergent throughout. However, it can be time-consuming and wasteful of detergent if the column must be re-equilibrated in a new detergent before each run is carried out. Furthermore, as the main purpose of detergent screening is to compare traces, the trends will remain in the traces even if the conditions are not ideal. Thus, a compromise can be reached whereby the protein is solubilized in the detergent of interest but the column is run in a standard buffer with a single detergent across all the runs (e.g., DDM)11, which can save time and detergent consumables.
By modifying the FLPC equipment used, the throughput of the FSEC protocol can be significantly increased, and the sample requirement can be minimized. For example, an FPLC or HPLC system could be equipped with an autosampler, a smaller bed volume analytical column (such as a 3.2 mL analytical SEC column), and an in-line fluorescent detector for monitoring continuous FSEC traces directly from the column. The resulting setup would allow more FSEC runs to be carried out in a shorter period of time and remove the manual plotting step, thus allowing a greater number of conditions to be tested in a shorter time frame. Furthermore, the sample requirement would be further reduced, as fewer samples would have to be prepared and loaded onto the FSEC column for each run. This would open up possibilities for reducing the expression cultures to a plate-based format, as such little material would be required for the analysis.
Strengths and weaknesses of the FSEC compared to other methods
A disadvantage of FSEC is that the membrane protein constructs need to be designed to introduce the fluorescent label, and on introduction, there is a small possibility that the placement of the label could interfere with the function or folding of the membrane protein of interest. In addition, the FSEC protocol, as described here, monitors the characteristics of a membrane protein in the presence of cell lysate, which is a crude mixture of proteins. The behavior of a membrane protein in this environment may be different than when the membrane protein of interest is subjected to a preparative SEC column at the end of purification when fully isolated from other proteins. Furthermore, FSEC provides a somewhat qualitative measure of protein quality. However, by converting the FSEC trace to a monodispersity index, as described in step 4.3.3 of the protocol, a quantitative measure of protein quality can be obtained.
FSEC is not the only method that can be used in the early analysis of membrane protein constructs, solubilization conditions, and purification buffer composition. The alternative approaches have both advantages and disadvantages over FSEC. For example, fluorophore-based thermostability assays exist, particularly the use of the dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM)16,17. The advantage of this method is that, unlike FSEC, which provides a qualitative measure of protein quality, thermostability assays provide a quantitative measure in the form of a relative melting temperature. Furthermore, there is no requirement to introduce a fluorescent tag on the protein construct. However, the disadvantages of thermostability assays compared with FSEC are that purified protein must be used and that the assay is not compatible with all protein constructs, as it relies on the advantageous positions of native cysteine residues in the folded protein.
Another method that has similarities to both FSEC and fluorophore-based thermostability assays is an assay that measures the temperature sensitivity of a membrane protein. In this assay, the protein is challenged with different temperatures, and the protein that remains in solution after centrifugation is detected. Detection in this method has been conducted in several ways, including measuring the fluorescence in solution18, the fluorescence of an SDS-PAGE gel band19, or the signal intensity in a western blot20. However, a significant disadvantage of these approaches is that the assay is very labor-intensive and prone to high noise in the results, as each individual temperature point must be collected independently.
Finally, several more advanced biophysical techniques can be used to assess membrane protein quality in a similar manner to FSEC, for example, flow-induced dispersion analysis21, microscale thermophoresis22, or SPR. Although very powerful approaches, the disadvantage of these methods is the requirement for highly specialized instruments to run the analyses.
In conclusion, FSEC provides an invaluable tool for use in membrane protein production campaigns, and although it is not the only option, it has several distinct advantages over other methods, as listed above. The cross-validation of the results by orthogonal assays is always recommended, and none of the methods discussed above are mutually exclusive of one another.
The authors have nothing to disclose.
We would like to acknowledge the help and support of the whole Peak Proteins team. From the cell science team, we would like to acknowledge Ian Hampton for his valuable insights and guidance in insect cell expression. We would like to thank Mark Abbott for providing the resources and opportunity to pursue this project.
1 mL and 5 mL plastic syringes | Generic | - | Syringes for transfer of samples |
10x EDTA Free Protease inhibitor cocktail | Abcam | ab201111 | Protease inhibitors |
15 mL tubes | Generic | - | 15 mL tubes for pellet preparation and solubilisation |
2 mL ultra-centrifuge tubes | Beckman Coulter | 344625 | Tubes for ultra-centrifuge rotor |
50 mL tubes | Generic | - | 50 mL tubes for cell harvest |
96 deep-well blocks | Greiner | 15922302 | For collecting 0.2 mL SEC fractions |
ÄKTA V9-L loop valve | Cytiva | 29011358 | 5 posiiton loop valve for the ÄKTA FPLC system |
ÄKTA F9-C fraction collector | Cytiva | 29027743 | 6 position plate fraction collector for the ÄKTA FPLC system |
ÄKTA pure 25 L | Cytiva | 29018224 | FPLC system for running the experiment |
Benchtop centrifuge (e.g. Fisherbrand GT4 3L) | Fisher Scientific | 15828722 | Centrifuge for low-speed spin |
Blunt end filling needles | Generic | - | For transfer of samples |
Bottle top vacuum filter | Corning | 10005490 | Bottle top vacuum filter for filtering SEC buffers |
Cholesteryl hemisuccinate (CHS) | Generon | CH210-5GM | Additive for detergent solubilisation |
Disposable multichannel reseviour | Generic | - | Resevior for addition of water or buffer to 96-well micro-plate |
Dodecyl maltoside (DDM) | Glycon | D97002-C-25g | Detergent for solubilisation |
eGFP protein standards | BioVision | K815-100 | eGFP standards for fluorescent calibration curve |
Glycerol | Thermo Scientific | 11443297 | Glycerol for buffer preparation |
HEPES | Thermo Scientific | 10411451 | HEPES for buffer preparation |
High molecular weight SEC calibration standards kit | Cytiva | 28403842 | Molecular weight calibration kit for SEC |
Lauryl maltose neopentylglycol (LMNG) | Generon | NB-19-0055-5G | Detergent for solubilisation |
Low molecular weight SEC calibration standards kit | Cytiva | 28403841 | Molecular weight calibration kit for SEC |
MLA-130 ultra-centrifuge rotor | Beckman Coulter | 367114 | Rotor for ultracentrifuge that fits 2 mL capacity tubes |
Opaque 96-well flat-bottom micro-plate | Corning | 10656853 | 96-well for reading fluorescent signal in plate reader |
Optima MAX-XP ultra-centrifuge | Beckman Coulter | 393315 | Centrifuge for high-speed spin |
pH meter | Generic | - | For adjusting the pH of buffers during preparation |
Prism | GraphPad | - | Graphing software for plotting traces |
Rotary mixer | Fisher Scientific | 12027144 | Mixer for end over end mixing in the cold |
Sodium chloride | Fisher Scientific | 10316943 | Sodium chloride for buffer preparation |
Sodium hydroxide | Fisher Scientific | 10488790 | Sodium hydroxide for buffer preparation |
Spectramax ID3 Plate Reader | Molecular Devices | 735-0391 | Micro-plate reader capable of reading fluorescence |
Stirrer plate | Generic | - | For stirring buffers during preparation |
Styrene maleic acid (SMA) | Orbiscope | SMALP 300 | Polymer for detergent free extraction |
Superdex 200 Increase 10/300 GL | Cytiva | 28990944 | SEC column for running the experiment. The bed volume of this column is 24 mL. The recommended flow rate for this column in 0.9 ml/min (in water at 4 °C). The maximum pressure limit for this column is 5 MPa. |
Vacuum pump | Sartorius | 16694-2-50-06 | For filtering and degassing buffers |