Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Summary

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Abstract

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Introduction

Numerous strategies are available for comprehending cellular biology. One approach involves a top-down strategy, wherein genetic mutations are introduced into a gene, followed by the evaluation of resulting phenotypic changes in a model organism. Conversely, a reductionist approach entails the initial elucidation of molecular mechanisms and enzymatic functions of a particular protein, accompanied by the characterization of its interactions with other cellular components. Subsequently, the impact of this protein on a biological pathway is assessed. Although each research approach possesses its inherent advantages and limitations, achieving a comprehensive understanding of a biological pathway necessitates interdisciplinary investigations.

With DNA being the genetic blueprint of life, understanding the mechanisms of DNA duplication and genome maintenance has been an area of active interest for over seven decades. Studies in the field of DNA replication have yielded copious data concerning the individual structures and functions of numerous replication proteins. These inquiries, which encompass mechanistic aspects and biochemical activity assays, have been made feasible through the purification of these proteins, enabling their meticulous examination in an in vitro milieu. Consequently, protein purification emerges as an indispensable and ubiquitous technique in the majority of research endeavors geared toward unraveling mechanistic insights into DNA replication.

This article presents a methodology for isolating a DNA replication protein tagged with 6X-histidine, which has been overexpressed in bacterial cells. The protein of interest is human flap endonuclease 1 (FEN1), a structure-specific nuclease that plays a pivotal role in lagging strand replication and is also a critical participant in DNA repair pathways like base excision repair (BER)^1,2,3. FEN1's primary function is to cleave at the base of a 5'-displaced flap structure, an intermediate that arises during DNA replication or BER. Initially, biochemical investigations assessing the enzymatic activity of FEN1 suggested a "tracking" mechanism, wherein the nuclease would recognize the free 5'-phosphate end of a flap structure and then follow along the flap to its base before cleaving it⁴. Subsequent research revealed that FEN1 operates via a "threading" mechanism, wherein it first binds to the base of the flap and then threads the free 5' end through its active site prior to cleavage (Figure 1)⁵. The ability to overexpress and isolate recombinant FEN1 has facilitated these breakthroughs, enabling researchers to employ it in biochemical and structural investigations.

Affinity chromatography is a commonly used separation method to purify DNA. This technique uses the reversible binding affinity of target proteins towards ligands immobilized on a resin to specifically trap the protein of interest. One of the most widely used bio-affinities is the robust interaction between the amino acid, histidine, and metal ions such as nickel and cobalt and hence, can be captured onto a resin charged with Ni²⁺ or Co²⁺.

The DNA sequence encoding a string of 6-9 histidine residues (His) is frequently incorporated into the plasmid construct which encodes the protein of interest (at either the N-terminus or C-terminus), tagging the protein with a 6X-His-tag or a poly-His tag. The His-tagged protein can then be easily purified by immobilized metal affinity chromatography (IMAC), a subtype of affinity chromatography whereby metal ions on the resin capture proteins with an affinity tag, which can later be eluted using appropriate elution agents. Transition metal ions such as Ni²⁺ and Co²⁺ can be immobilized onto agarose or silica gel matrices derived from N,N,N'-tris-(carboxymethyl)-ethylenediamine or nitrilotriacetic acid (NTA) groups⁶.

Metal ligands are known to be robust against degradation by physical, chemical, and biological factors and hold this advantage over other types of ligands^6,7. Additionally, the His-tag is a relatively small tag and does not significantly impact protein structure or function⁷. However, in a bacterial expression system, many chromosomally expressed proteins have an affinity towards metal ions and may co-purify with the target protein. Nickel and cobalt are the typical metal ions used in IMAC matrices. The Ni-NTA resin and the TALON cobalt-based resin are commonly used for the purification of His-tagged proteins.

Ni-NTA versus TALON
The respective metal ions of both Ni-NTA and TALON are immobilized on the resin through NTA ligands. Ni-NTA is thought to have a higher binding capacity, binding up to 100 mg/mL of protein. This can result in a higher protein yield, with the caveat that contaminant proteins may be co-purified. In contrast, the resin has a higher binding specificity towards His-tagged proteins and may be able to produce fractions of higher purity. In this study, we aim to compare the purification efficiency of both resins using the referenced automated fast protein liquid chromatography system, the NGC system (see the Table of Materials).

Buffers and compatibility
Buffers are required during protein purification for cell lysis, sample preparation, resin equilibration, and elution of the captured protein from the resin. Tris, MOPS, and HEPES buffers up to a concentration of 100 mM are the known compatible buffers for the Ni-NTA resin. Buffers often include reducing agents to prevent the oxidation of protein and protein aggregation. However, above a threshold limit, reducing agents could strip the resin of metal ions. The recommended concentration of reducing agents such as beta-mercaptoethanol (BME) or dithiothreitol (DTT) is below 1 mM for the above-mentioned nickel- and cobalt-based resins.

The buffers for the purification of hFEN1 are Tris buffers containing NaCl, BME, phenylmethylsulfonyl fluoride (PMSF), EDTA, and glycerol. NaCl maintains the protein in soluble form and disrupts molecular interactions such as DNA binding. BME reduces oxidized proteins and thereby prevents protein aggregation. PMSF) is a protease inhibitor that prevents protease-mediated degradation of target protein. EDTA eliminates divalent cations from the sample, preventing their access to nucleases and proteases. Glycerol enhances the stability of the protein in aqueous form. Additionally, the lysis buffer contains complete protease inhibitor tablets to ensure maximum protection of target protein from degrading proteases during cell lysis. The equilibration and elution buffers contain imidazole, with the elution buffer containing higher quantities for the imidazole to displace the bound protein from the resin during elution.

Next Generation Chromatography (NGC) system
This automated, medium-pressure chromatography system designed for fast-flow protein liquid chromatography (FPLC) uses two pumps to simultaneously pump two different buffers and is capable of injecting a wide range of sample volumes from 250 µL to 100 mL. The sample loop (known as the Dynaloop in this system), makes it possible to inject larger sample volumes. The system can be operated using the Chromlab software, which facilitates customized method creation, manipulation of purification runs, and analysis of UV peaks and protein fractions.

Protocol

1. Sample preparation

1. To purify recombinant FEN1, express the construct (pET-FCH-FEN1) in BL21(DE3) cells as previously described by Ononye et al.⁸.
   1. Inoculate LB media (Table 1) with 1% overnight culture and grow the cells at 37 °C until the OD reaches 0.6.
   2. Induce with 0.4 M isopropyl-beta-D-thiogalactoside (IPTG) and grow the culture for an additional 3 h.
   3. Harvest the cells by centrifuging the culture at 5,000 × g for 15 min at 4 °C. Store the pellet in a -80°C freezer.
   4. On the day of the purification, remove the cell pellet from the -80 °C freezer, allow it to thaw on ice, and resuspend the pellet in 100 mL of lysis buffer (Table 1).
      NOTE: The cell lysate should always be placed on ice during handling to prevent proteolysis. Once thawed, the entire purification process must be completed on the same day. The cell lysate should not be stored in the refrigerator overnight.
2. Transfer the suspension to a 250 mL beaker and sonicate on ice with 30 s ON and 30 s OFF at 35% amplitude for 15 min.
   NOTE: Consult the manufacturer's settings to identify the amplitude.
3. Transfer the sonicated sample to a centrifuge tube and spin at 27,000 × g for 30 min at 4 °C to obtain a clear lysate. Place the cell lysate on ice in the 4 °C refrigerator until sample injection.

2. Preparing affinity columns

1. Fit the columns (one for the Ni-NTA resin and one for the cobalt-based resin) to a clamp stand such that the columns are held as straight as possible, avoiding any slanting of the columns. Ensure that the bottom of the columns are plugged in with a stopper and add 1-2 mL of starting buffer to the bottom of each column to prevent air bubbles during column packing.
2. Shake the resin bottles gently until the resin is fully resuspended. Open the top of the columns and pour in 10 mL of each slurry in a continuous manner to avoid the inclusion of air bubbles. Pour the resin against a metal spatula or glass rod held at the rim of the column to avoid air bubbles in the column.

3. Column packing using a flow adaptor

1. Unplug the stopper from the column and connect the bottom to one end of the tubing. Connect the other end of the tubing to the UV port of the FPLC system.
2. Ensure that the bed support housing has been pulled into the flow adaptor body and slide the flow adaptor body onto the top of the column (Figure 2A). With the cam latch in position 1, lower the flow adaptor taking care not to insert it into the buffer (Figure 2B). Switch the cam latch to position 2 (Figure 2B). Connect the tubing to the system and start the flow of the starting buffer for 1-2 min to rid the tubing of air and prevent air bubbles during packing.
3. Switch the latch back to position 1 and lower the flow adaptor into the buffer in one smooth motion, avoiding the inclusion of air bubbles, and switch the latch back to position 2. This allows for some buffer to enter the flow adaptor and removes any remaining bubbles.
4. Switch the latch to position 3 and tighten the cam base and locking ring (Figure 2B).
5. Start the flow of buffer at 10 mL/min to pack the column. Continue this step for 3 min or until the resin has been well packed.

4. Priming the sample loop of the FPLC system

1. Connect the tubing from the top of the sample loop to Loop E on the FPLC system (Figure 3).
2. Connect the tubing from the bottom of the sample loop to Waste 2 on the FPLC system.
3. Insert one end of the tubing into the column port of the FPLC system and place the other end in the waste bottle.
4. Insert the tubing from pump A in filtered double distilled water (ddH₂O).
5. Double-click on the pump settings using the software (Figure 4A, labeled 2) and select system pump inject loop (Figure 4C).
6. Click on the flow rate settings (Figure 4A, labeled 1) and change the flow rate to 10 mL/min. Enter 0% in the %B box (Figure 4B).
7. Start the flow to flush the sample loop with water, which will push the sliding seal downwards.
8. Once the sliding seal reaches the bottom of the sample loop, stop the flow. Switch pump A from ddH₂O to the starting buffer. Connect the tubing from the top of the sample loop to Waste 2 and that from the bottom to Loop E. Leave the tubing from the column port in the waste.
9. Start the flow at 10 mL/min. Once the sliding seal pushed upwards by the incoming starting buffer reaches the top of the sample loop, stop the flow. The sample loop is now ready for sample injection.

5. Sample injection

1. Double-click on the pump settings and select Manual load loop (Figure 4C).
2. Unscrew the tubing from Loop E and place it in the waste container.
3. Connect the tubing from the column port to the tubing on top of the flow adaptor.
4. Draw up the sample using a 30 mL syringe; then, attach the syringe to the injection port adaptor.
5. Screw the syringe into the injection port and inject the sample, ensuring that it is visibly injected into the sample loop. Store 100 µL of the sample (label input lysate) in a microcentrifuge tube at -20 °C for SDS-PAGE analysis
   NOTE: Depending on the total sample volume, 2-3 injections may be required.
6. Plug the injection port with the column cap.
7. Connect the tubing from the bottom of the sample loop back into the Loop E port.
8. Place the tubing from pump B in the elution buffer.

6. Method creation and analysis using the FPLC system-linked software

1. Navigate to the Home tab and select New method.
2. Select the Method settings tab on the left column, navigate to the Column type dropdown menu, and select Custom (Figure 5).
3. Enter the column volume (5 mL).
4. Enter the required flow rate for the run (1 mL/min).
5. Navigate to Unit selection and select CV (column volumes) as the method base unit.
6. Ensure that Inlet A is set to Buffer A and Inlet B is set to Buffer B (the default setting).
7. Select the Method Outline tab on the left side menu. Add the steps of the purification in the following order: equilibration, sample application, column wash, and elution.
   1. Under equilibration, set the equilibration volume to 10 CV. Ensure that the buffer setting is kept at 0% B.
   2. Under the sample application tab, set the sample volume by observing the sample loop. The position of the sliding seal indicates the sample volume.
   3. Under column wash, set the wash volume to 5 CV. Disable fraction collection for this step; collect the wash as a whole in a beaker during the run, rather than collecting as fractions.
   4. For elution, set the gradient volume to 14 CV starting from 0% Buffer B to 100% Buffer B.
8. Save the method and start the run.
9. Ensure that the fraction collector starts at the position of the first fraction.
10. Enter the run name and click Start.
11. During the equilibration phase, monitor the real-time UV curve. Ensure that all connections are secure with no leakage; confirm a complete circuit by looking for the buffer flowing into the waste container. If the UV curve has not stabilized during the last minutes of equilibration, extend the equilibration step until stabilization is achieved (Figure 6) .
12. At the time of sample application, switch the tubes going into the waste container to a clean beaker. During this step, collect the unbound proteins flowing out of the waste tubes as the flowthrough (label as FT).
13. Look for a sharp increase in the UV curve that likely reaches the maximum possible reading of 3,000 maU (Figure 6).
    NOTE: A typical curve plateaus at 3,000 maU through most of the sample application step.
14. During column wash, as residual unbound proteins are being washed off with only bound proteins remaining on the resin, look for a sharp drop in the UV curve (Figure 6). If the UV curve has not dropped by the end of the wash step, extend the step by clicking hold step until the UV has dropped sufficiently. At the beginning of this step, transfer the waste tubes from the FT beaker to another clean beaker labeled Wash.
15. When the elution step starts, ensure that the fraction collector moves into position and begin collecting fractions. Visualize the elution of protein by looking for peaks in the UV curve (Figure 6).
16. After run completion, navigate to the Home tab and select Open Run. Open the run to view the chromatogram and select peak integration on the top menu to mark the peaks obtained during the run.
17. Select the Fractions tab to visualize the fractions containing eluted protein.

7. SDS PAGE analysis

1. Mix 15 µL of each fraction containing protein with 5 µL of SDS sample loading buffer (Table 1). Heat the samples at 95 °C for 5 min.
2. Load samples into precast gels and run at 160 V for 45 min along with prestained protein marker.
3. Stain the gels in Coomassie brilliant blue staining solution for 30 min (Table 1).
4. Wash the gel in ddH₂O (repeat 3x).
5. Destain the gel in destaining solution for 1 h (Table 1).
6. Observe protein bands; detect FEN1 at the 42 kDa mark.
7. Pool fractions containing FEN1 and concentrate the pool using a 10 kDa centrifugal filter. Aliquot and store in the -80˚ °C freezer.

Results

BL21 (DE3) cell lysates expressing hFEN1 were passed through equilibrated Ni-NTA and TALON resins. The Ni-NTA resin is charged with Ni²⁺ ions and has a high binding capacity. The results show that the Ni-NTA resin yields a higher quantity of FEN1 compared to the TALON resin (Figure 7). The Ni-NTA resin is also known to non-specifically bind to other chromosomally expressed proteins. Cell lysate passed through the cobalt-based resin, was purified with high purity but a lower yield,...

Discussion

Affinity chromatography is a widely used technique to purify DNA-binding proteins. Immobilized metal affinity chromatography (IMAC) is a specific type of affinity chromatography that uses metal ions to capture the histidine residues of a peptide sequence. This is why the "6X-His tag" or "poly-His tag" is attached to the N-terminus or the C-terminus of proteins to be purified. Nickel and cobalt are the most commonly used metal ions and vary in their compatibility with reagents such as BME and DTT normally ...

Materials

Name	Company	Catalog Number	Comments
4x Laemmli sample buffer	BioRad	1610747
Acetic acid	Merck	UN2789
Beta-mercaptoethanol (BME)	Sigma	M-6250
Chromlab software version 6.1.27.0	BioRad		operates the NGC system
Complete MINI protease inhibitor tablet	Roche	11836153001
Coomassie Brilliant Blue R	Sigma	B0149
Dithiothreitol (DTT)	Dot Scientific	DSD11000
Econo-Column glass	BioRad	7371512
Ethylene diamine tetraacetic acid (EDTA)	Dot Scientific	DSE57020
Flow adaptor	BioRad	7380014
Glycerol	Dot Scientific	DSG22020
Imidazole	Dot Scientific	DSI52000
Methanol	Fisher Scientific	A412
Mini PROTEAN TGX gels	BioRad	4561084
NGC Chromatography System	BioRad		automated liquid chromatography system
Ni-NTA Agarose	Qiagen	1018244
Phenyl-methyl-sulfonyl fluoride	Dot Scientific	DSP20270
PreScission Plus Protein Dual Color Standards	BioRad	1610374
Sodium chloride	Dot Scientific	DSS23020
TALON metal affinity resin	Takara	635502
Tris Base		DST60040