Protein Methods Articles:
Thermo Fisher Scientific, Rockford, IL
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Performance characterization of Pierce Glutathione Superflow Agarose
Purity, dynamic binding capacity, reusability and chemical compatibility properties of a glutathione resin for FPLC purification of GST tagged fusion proteins.
Aaron McBride, M.S.;
Ramesh Ganapathy, Ph.D.;
Scott Meier, M.S.;
Barb Kaboord, Ph.D.;
Paul Haney, Ph.D.;
The study of protein function and structure has been greatly enhanced by recombinant protein expression strategies that incorporate fusions of affinity tags to aid in purification. The glutathione S-transferase (GST) tag is particularly popular for two primary reasons: (1) the GST tag usually increases the solubility of proteins to which it is fused, thereby aiding in their expression and production, and (2) purification of GST-tagged proteins using glutathione (GSH) as the ligand typically yields greater than 95% pure protein.
A variety of glutathione resins are commercially available, differing in multiple aspects such as the resin support and the immobilization strategy of the glutathione. These differences can affect binding capacity, reusability, flow characteristics, and chemical compatibility of each glutathione resin, ultimately determining the quality of results (yield, purity, speed) in particular applications. Careful selection and method optimization may be required based on the specific application and scale.
Thermo Scientific Pierce products include several glutathione-based supports for GST-tagged protein purification; these include magnetic beads, two kinds of agarose resin, and various sizes and formats. Pierce Glutathione Superflow Agarose uses glutathione immobilized onto highly crosslinked (Superflow) 6% beaded agarose. In this article, we detail performance characteristics of Pierce Glutathione Superflow Agarose, which is especially suited for small- and large-scale purifications of recombinant GST tagged proteins by FPLC.
RESULTS and DISCUSSION:
To demonstrate purification efficiency of Pierce Glutathione Superflow Agarose, we over-expressed and purified several recombinant GST-tagged fusion proteins by FPLC: human rhinovirus type 3C protease (HRV 3C), SH2 domain of tyrosine-protein kinase Syk (Syk), and growth arrest and DNA damage inducible protein (GADD34). Purifications resulted in elution fractions with 85 to 98% purity levels, as assessed by SDS-PAGE and densitometry analysis (Figure 1). We also tested and compared purification performance between Pierce Glutathione Superflow Agarose and an alternative source of glutathione resin (Figure2). Similar high levels of purity were rapidly and easily achieved with both resins.
Figure 1. Purification of GST-tagged fusion proteins using Thermo Scientific Pierce Glutathione Superflow Agarose. Clarified over-expressed E. coli lysates were loaded onto packed columns packed with Pierce Glutathione Superflow Agarose. Unbound protein was removed with wash buffer. Bound protein was then recovered using elution buffer (see Methods). Pooled fractions of load lysate (L), flow-through (F), wash (W) and eluate (E) were separated by SDS-PAGE and stained with Thermo Scientific Imperial Stain (Part No. 26415). Purity was determined by densitometry analysis of the stained eluate lanes with Thermo Scientific myImageAnalysis Software (Part No. 62237).
Figure 2. Large-scale FPLC purification of GST produces >98% purity of target protein. Biomass (170g) containing overexpressed GST was lysed with 1.7L of lysis buffer and then 0.75L of lysate was loaded onto equilibrated 200mL columns (50mm x 100mm) of Pierce Glutathione Superflow Agarose (left) or Glutathione Sepharose™ 4 Fast Flow (GE Healthcare, Cat. 17-5132-03, right) at a linear flow rate of 30cm/hour. Columns were washed with binding buffer until the UV280 reached baseline; then bound protein was eluted with elution buffer, and fractions containing purified GST were pooled. Aliquots of load, flow-through, wash, and eluate fractions were separated by SDS-PAGE, stained with Thermo Scientific Imperial Protein Stain (Part No. 24615) and evaluated using Thermo Scientific myImageAnalysis Software (Part No. 62237) to determine purity. Total yield, recovery, and purity were similar for both resins.
Dynamic Binding Capacity
Maximizing the performance of protein purification in FPLC protocols is critical and requires a good understanding of the binding characteristic of a resin under a variety of flow rates. The longer the protein sample is in contact with the resin, the higher the binding capacity will be until it reaches the theoretical maximum capacity for the support (Figure 3). Greater binding capacities are possible with slower flow rates, but these conditions necessary require increased processing time. Therefore, the profile of a resin’s dynamic binding capacity is an important consideration when optimizing a purification process.
Dynamic binding capacity of a column run at a given specific flow rate (a value that is inversely proportional to residence time) is typically reported as the amount of protein that will bind to the resin before 10% of the target protein accumulates in the flow-through fraction (called the “breakthrough”). Maximizing dynamic binding capacity enables the use of higher flow rates and less resin, while still minimizing target protein loss and controlling process time.
We determined a profile of dynamic binding capacities for Pierce Glutathione Superflow Agarose by applying highly purified GST to a 1mL resin bed at several different flow rates (Figure 3). At a flow rate of 0.5mL/min (equivalent to a 2-minute residence time), the resin exhibited a binding capacity of nearly 10mg of GST per mL of packed resin. At a slow flow rate of 0.1mL/min (equal to a 10min residence time), the resin exhibited a binding capacity of approximately 24mg of GST per mL of packed resin.
Variations in tag accessibility between recombinant protein, as well as the presence of other proteins and biological molecules in a complex lysate may affect the resin’s overall binding capacity. Therefore, it is important to determine the appropriate balance between flow rate (production run speed) and capacity (production yield) for each process being developed.
Figure 3. Dynamic binding capacity vs. flow rate for Pierce Glutathione Superflow Agarose. Five columns (diameter = 0.5cm) packed with 1mL of Pierce Glutathione Superflow Agarose were loaded with purified GST (1mg/mL) at flow rates of 1.0, 0.5, 0.3, 0.2, and 0.1/min (corresponding to residence times of 1, 2, 3.3, 5 and 10min, respectively). For each column, the dynamic binding capacity (total protein loaded) was determined at 10% breakthrough (i.e., the point at which the instantaneous flow-through absorption at 280nm corresponds to 0.1mg/mL GST).
Reusability and Compatibility
Depending upon the laboratory setting, affinity purification may be performed at scale ranging from sub-milliliter volumes to many liters. Although it is often easier and cleaner to pack and use a new column each time for small-scale purification, this is not practical for large-scale processes involving repeated purification of same target protein. Pierce Glutathione Superflow Agarose is a robust highly crosslinked resin designed for use at a wide range of scales. Superflow resin can withstand linear flow rates as high as 1260cm/hr without compressing. By contrast, Sepharose™ 6B (agarose) typically begins to compress at approximately 400cm/hr (data not shown).
To demonstrate the reusability of Pierce Glutathione Superflow Agarose, we evaluated the profiles of repeated cycles of chromatography on a single column (Figure 4). We challenged the resin with an E. coli lysate containing overexpressed GST-Syk, then performed five clean-in-place (CIP) cycles using 6M guanidine and 70% ethanol. We repeated this pattern of 1 challenge plus 5 CIP-treatments, resulting in 26 cycles containing 6 rounds of actual purification. No significant differences were observed in the graphed profiles (UV280nm traces, Figure 4A) or stained band profiles (Figure 4B), indicating no decline in yield or purity.
Pierce Glutathione Superflow Agarose has low non-specific binding, and very little residual protein remain on the column after purification. In most cases, simply washing the column with wash buffer to eliminate any excess glutathione is all that is necessary for regeneration; however, depending upon the clarity, solubility and quality of the sample or cell lysate, precipitated proteins and other hydrophobic substances can accumulate on the resin over time. The buildup of proteins can lead to an increase in column back-pressure and decreases in protein purity and yield during purification. If this situation does occur, resin performance can be restored with a clean-in-place (CIP) protocol: wash with 6M guanidine-HCl and 70% ethanol.
Figure 4. Reuse of Pierce Glutathione Superflow Agarose using a standard clean-in-place procedure. A single, 1mL column (column diameter = 0.5cm) of Pierce Glutathione Superflow Agarose was processed through 26 purification cycles. The column was challenged with an E. Coli lysate containing overexpressed GST-Syk every 5th cycle and mock purifications were performed in all the other cycles. All cycles included a clean-in-place procedure that included a 2-column volume (CV) wash with 6M guanidine, 5-CV wash with wash buffer, 4-CV wash with 70% ethanol, and an additional 5-CV wash with wash buffer. (Top) The chromatograms show the UV280 traces were similar between runs, when the column was challenged with lysate and (bottom) yields and purity were similar in all purifications.
Finally, we evaluated the compatibility of Pierce Glutathione Superflow Agarose with various chemicals used during purification, cleaning or storage of chromatography supports (Figure 5). We stored the resin as a 50% slurry in the indicated solutions for an extended period of time (2 hours or 1 week). Following incubation, we equilibrated each resin sample in binding buffer and compared its static batch-binding capacity to the untreated resin sample. None of these chemicals significantly affected binding capacity. More stringent clean-in-place protocols using NaOH (pH > 12) did dramatically decrease the binding capacity of the resin (data not shown) and should be avoided.
Figure 5. Chemical compatibility of Pierce Glutathione Superflow Agarose. 1mL aliquots of Pierce Glutathione Superflow Agarose were stored as 50% slurries in various solutions and then used for purification. Following incubation in each solution, duplicate 50µL samples of each resin-aliquot were added to 2mL spin columns (for subsequent processing at 700 x g), washed 3 times with 1mL wash buffer and then incubated 4 x 30 minutes with 0.625mg (for a total of 2.5mg) of GST by end-over-end mixing at 22°C. After the final centrifugation to remove the protein solution, columns were washed 3 times with 0.5mL wash buffer; finally, bound protein was eluted with 3 x 0.5mL aliquots of elution buffer. Yield in the combined elution fraction of each sample were determined in triplicate by 280nm assay. Error bars are standard deviations of the duplicate samples of each condition.
All purifications and binding experiments were performed using the following buffer conditions, unless noted otherwise:
- Binding/wash buffer (50mM Tris-HCl pH 8.0 and 125mM NaCl)
- Elution buffer (50mM Tris-HCl pH 8.0, 125mM NaCl, 10mM reduced glutathione)
All wash and elution steps were done with 5 to 15 column volumes (CV). The results outlined in this article are considered typical; however, protein yield and purity are influenced strongly by recombinant fusion protein expression levels, solubility, and fusion tag accessibility. In some cases optimization of the buffer conditions and flow rates may be required.