1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University
Murphy, P. J. M., Stone, O. J., Anderson, M. E. Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification. J. Vis. Exp. (55), e3060, doi:10.3791/3060 (2011).
In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.
1. Buffer and Sample Preparation
2. Physical Setup and Plumbing of DuoFlow Chromatography System
3. Priming System Lines, Programming Run Method, and Equilibrating HIC Columns
4. Sample Loading
5. Programming the Software Method and Running the Column Scouting Protocol
6. Representative Results:
Representative HIC salt gradient, conductivity, and column pressure of the scouting runs are presented in Figure 4. The change in salt concentration (blue line), as measured by the percentage of buffer drawn from high-salt buffer lines is typical of HIC methodology. As the salt concentration decreases, proteins bound to the column elute. Conductivity (red line), which corresponds to observed salt concentration, is measured in-line immediately following the QuadTec and UV detectors. The off-set between salt gradient and conductivity tracings indicates the time required for buffer to travel from the buffer inlet, through the system, and to the conductivity monitor. Throughout the sample run, the system pressure (grey lines) and pH remain relatively constant.
Figure 5 shows chromatograms of sequential HIC column scouting runs. The in-line detection of total protein (A280, blue line) and GFP (A397, green line) is accomplished by measuring the absorbance of light at 280 nm and 397 nm, respectively. It is possible to approximate the relative GFP abundance and separation for each scouting run by comparing the two lines. GFP bound to the tested HIC columns with different degrees of affinity and its elution profile varied. Selection of a preferred HIC column for future purifications is based on identifying the column that produces the sharpest GFP elution peak and greatest separation from other proteins.
In Figure 6, culture tubes for Fractions 10, 12, 14, 16, and 18 of the Phenyl FF (high sub) scouting run were visualized under ambient room (fluorescent) light and ultraviolet (UV) light. Tubes were viewed both face-forward (left panels) and top-down (right panels) in order to observe the characteristic GFP emission spectrum. GFP (green) is clearly detected in Fraction 14 in both UV images. These post-run data correspond nicely with the in-line detection of GFP by measuring eluate absorbance at 397 nm (A397, green line), which also identifies Fraction 14 as containing the major peak of eluted GFP. The diffuse blue in the left UV panel is light emitted from the UV lamp.
Figure 1. Schematic representation of this protocol. Bacterial lysate containing protein of interest (GFP, target protein) is prepared, diluted into a high-salt buffer equivalent to the chromatography start buffer, and filtered. Once the liquid chromatography system is prepared, sample is loaded and the target protein is separated from other proteins contained in the lysate using a hydrophobic chromatography medium contained in a pre-packed HIC column. The separation method is repeated several times using different chromatography media (referred to as column scouting) in order to determine which media provides the best GFP separation. The eluted proteins (eluate) is analyzed in-line using detectors included in the chromatography system and it is collected into smaller fractions for subsequent (post-run) analysis. Based on the in-line and post-run analysis of GFP activity and separation, an optimum HIC column and chromatographic medium are identified.
Table 1. Key chromatography system components used in this protocol.
Figure 2. Diagram of Bio-Rad DuoFlow medium-pressure liquid chromatography system employed. Key features of the system include automated buffer blending, loading of sample for sequential column runs, automated selection of different chromatography columns, in-line analysis of the eluate, and tandem collection of the fractionated eluate. See text and Table 1 for details.
Table 2. Biophysical characteristics of HIC media tested. The name of the HiTrap HIC column is indicative of the ligand, ligand density, and bead size.
*FF = fast flow; HP = high performance. Larger bead size increases ligand binding capacity and flow rate. Smaller bead size increases chromatographic resolution. Information derived from literature provided by the manufacturer.
Figure 3. Connection of GE Healthcare HiTrap HIC column to Bio-Rad DuoFlow system.To the upstream 1/4-28 Delrin nut and ferrule (A1), a male Luer-to-female 1/4-28 fitting (B1) and male 1/16"-to-female Luer fitting (C) are connected. The fittings are attach to the HiTrap column after being flushed with buffer and having all bubbles removed. The column outlet is connected to a female 1/16"-to-male M6 fitting (D), male Luer-to female M6 fitting (E), and female Luer-to-female 1/4-28 fitting (B2). This entire assembly is connected to the downstream 1/4-28 Delrin nut and ferrule (A2). The upper panel shows the fittings separated and the lower panel shows the fittings assembled.
|Step #||Volume (mL)||Description||Parameters|
|1||0.00||Collect 1.0 mL fractions during entire run|
|2||0.00||Switch Columns||HIC Column 1
|3||0.00||Isocratic Flow||pH: 6.80
|Volume: 2.00 mL
Flow: 1.00 mL/min
|5||2.00||Zero Baseline||UV Detector|
|6||2.00||Inject Sample||Sample Load
|Auto Inject Valve
Volume: 0.50 mL
Flow: 1.00 mL/min
|7||3.00||Isocratic Flow||pH: 6.80
|Volume: 5.00 mL
Flow: 1.00 mL/min
|8||8.00||Linear Gradient||pH: 6.80
100%B -> 0%B
|Volume: 10.00 mL
Flow: 1.00 mL/min
|9||18.00||Isocratic Flow||pH: 6.80
|Volume: 3.00 mL
Flow: 1.00 mL/min
|10||21.00||Isocratic Flow||pH: 6.80
|Volume: 5.00 mL
Flow: 1.00 mL/min
|End||26.00||End of Protocol||Automatically Repeats with 5 additional HIC columns
|Scout type||Number of Runs: 6||Number of Steps Scouted: 1|
Table 3. BioLogic software protocol for HIC scouting method employed.
Figure 4. Representative HIC salt gradient, conductivity, and column pressure. As the salt concentration (blue line) decreases, conductivity (red line) does as well. The off-set between salt gradient and conductivity tracings indicates time required for buffer to travel from the buffer inlet to the conductivity monitor. System pressure (grey lines) remains relatively constant for the duration of the scouting run.
Figure 5. Compiled chromatograms of sequential HiTrap HIC column scouting runs. The in-line detection of total protein (A280, blue line) and GFP (A397, green line) is accomplished by measuring the absorbance of light at 280 nm and 397 nm, respectively. In this series of experiments, the sharpest GFP elution peak was observed with the Phenyl FF (high sub) column. The Phenyl FF (high sub) column also appeared to provide the greatest separation between GFP and other proteins. Additional 1 ml HiTrap HIC columns tested included Phenyl fast flow low substitution (Phenyl FF (low sub)), butyl fast flow (butyl FF), butyl-S fast flow (butyl-S FF), and octyl fast flow (octyl FF).
Figure 6. Representative post-run visualization of GFP in sample eluate. Eluate fractions were collected at a rate of 1/minute, and each was analyzed for GFP content. In this representative figure, culture tubes for Fractions 10, 12, 14, 16, and 18 of the Phenyl FF (high sub) scouting run were visualized under ambient room (fluorescent) light and ultraviolet (UV) light. Tubes were viewed both face-forward (left panels) and top-down (right panels). The diffuse blue in the left UV panel is light emitted from the UV lamp. GFP (green) is clearly detected in Fraction 14 in both UV images. The upper panel indicates the total protein (A280, blue line) and GFP (A397, green line) chromatogram tracings of the 5 fractions being visualized.
Liquid chromatography techniques have proven invaluable for preparing highly purified proteins necessary for conducting immunological 6, biochemical 7, and structural 8 studies. HIC purification methods most often require empirical determination of a preferred medium, and ligand structure, ligand density, and matrix bead properties all have been shown to impact chromatographic results 2, 3. Automated column scouting is an efficient approach for selecting a HIC medium for subsequent optimization and protein purification 4. The automated column scouting method presented can be readily adapted to various HIC protein purification strategies. Alterations in salt concentration, choice of salt, salt gradient, and pH may further improve purification conditions, and the effects of varying these essential parameters have been previously reviewed 13,14. HIC eluate containing a partially purified target protein can be further purified using a complementary chromatographic technique, such as ion exchange chromatography (IEX) or gel filtration/size exclusion chromatography 9,10.
Because of its unique light absorbance and emission characteristics, the elution profile of GFP can be identified using in-line and post-run approaches. To that end, this protocol can further be adapted for purification of recombinant GFP-fusion proteins, in which an unrelated target protein is “tagged” with GFP. GFP-tagged target proteins can be detected with UV light 11 and purified using various chromatographic approaches, including the HIC purification strategy described above. GFP purification has also become a pedagogical staple in biochemistry labs for the teaching of modern protein science techniques 12.
While basic HIC protein purification can be conducted using a substantially less robust chromatography system, the instrumentation presented here has a number of distinct advantages to aid in obtaining favorable results. Notable benefits of utilizing a highly automated system include improved run-to-run condition reproducibility, time-savings, and decreased opportunities for air to be introduced into the system 10. System-controlled buffer blending, which is facilitated by the system maximizer, allows for increased consistency in buffer preparation and experimental reproducibility. Drawing enough sample load into the dynamic sample loop for all scouting runs further ensures uniformity of the sample being added to each column and allows for sequential runs without interruption or manual reloading. Controlled sample injection from the sample loop onto the column decreases variability that may occur with manual sample loading. A pair of column selection valves allow for consecutive sample runs, each using a different HIC column, without having to replumb the system. Performing multi-wavelength analysis is particularly beneficial when assaying a protein with a unique spectrophotometric profile. In addition to GFP, cytochromes, flavoproteins, and other heme-containing proteins may benefit from this technique. Conductivity and pH monitoring devices allow for verification of real-time experimental conditions. Split fraction eluate collection allows for improved fraction handling and enables easy transfer for post-run analysis methods that require a minimal sample volume (e.g. ELISA, SDS-PAGE, western blot, and Experion microfluidic electrophoresis). While operating the second fraction collector offline requires manual synchronization, it allows for maximum flexibility in fraction collector selection. The most significant drawbacks to utilizing such a robust chromatography system for HIC column scouting and protein purification include the initial time and budgetary expenditures associated with instrument acquisition and operator training.
The protocol presented here utilizes a Bio-Rad DuoFlow chromatography system; however, equally robust instrumentation from other manufacturers, such as the ÄKTA Avant from GE Healthcare, may also be utilized and are capable of producing equivalent results. Even comparable chromatography system have unique characteristics (e.g. method programming, component nomenclature, operator preference, and scalability limitations) that should be considered before initiating a purification procedure or instrument acquisition.
Select chromatography reagents and supplemental instrumentation were provided by Bio-Rad.
This work was funded by National Institutes of Health grant GM086822 and National Science Foundation major research instrumentation grant DBI-0960313. The authors would like to thank Drs. Jon Miyake & Donna Hardy (Bio-Rad) and Jennifer Loertscher (Seattle University) for their technical expertise. Select chromatography reagents and supplemental instrumentation were generously provided by Bio-Rad.
|BioLogic DuoFlow Pathfinder 20 System||Bio-Rad||7602257||Includes system maximizer, mixer, F10 workstation, AVR7-3 valve, QuadTec UV/Vis detector with flow cell, BioFrac fraction collector, BioLogic software, and starter kit|
|AVR9-8 stream-select valve||Bio-Rad||7600408|
|Diverter valve SV3-2||Bio-Rad||7500410|
|Stream splitter valve||Bio-Rad||7410017|
|DynaLoop 25 Kit||Bio-Rad||7500451|
|BioFrac Fraction Collector||Bio-Rad||7410002||Two fraction collectors used|
|BioFrac microplate drop head adapter||Bio-Rad||74010088|
|BioFrac microtiter plate adapter||Bio-Rad||7410017|
|UV Optics Module||Bio-Rad||7500202|
|Econo Gradient Pump||Bio-Rad||7319001|
|Experion Pro260 Analysis Kit||Bio-Rad||7007102|
|HiTrap HIC column selection kit||GE Healthcare||28-4110-07||Includes 7 x 1 ml pre-packed columns of HIC media for scouting|
|GE Healthcare-to-Bio-Rad column fittings||GE Healthcare||18111251 and 18111257|