Pall Corporation (PLL): Filtration, Separations, Purification & Environmental Technology
Check OutLab Store
My AccountQuick Order
Shopping Cart

IMAC Purification of Polyhistidine-tagged Protein Using the AcroPrep™ 96 Filter Plate

Abstract

Bottlenecks in protein sample preparation highlight the need to optimize screening strategies and develop more effective automated systems to recover proteins for proteomic analysis. Fully automated purification strategies may include a number of steps where lysate clearance (AcroPrep 96 prefiltration plates), desalting and protein concentration (AcroPrep 96 ultrafiltration plates) are needed. Beyond standard sample preparation steps, optimizing the purification for over-expressed tagged-recombinant proteins requires the screening of a collection of subclones that often have highly variable expression and activity levels. Although size-separation applications can be done effectively using ultrafiltration, a more specific affinity Immobilized Metal Affinity Chromatography (IMAC) system is typically needed to purify biomolecules from crude lysates. This study demonstrates the use of AcroPrep 96 filter plates containing microporous membranes for the preparation of recombinant proteins isolated from IMAC resin minicolumns. The versatility of the AcroPrep 96 filter plate is demonstrated by the creation of a matrix of IMAC conditions within a single plate. The ability to create a screening matrix assists in the selection of an effective ligand for a target protein and allows the design of parallel processing tests to optimize resin to lysate ratios for a particular clone. The construction of the AcroPrep 96 filter plate allows both manual and automated handling, demonstrates bead retention, and shows consistent well-to-well performance, effectively giving the protein biochemist an edge in the development of protein purification protocols.

Top

Background

Immobilized Metal Chelating Chromatography
Immobilized Metal Affinity Chromatography (IMAC) (1,2) is a robust method for purifying polyhistidine-tagged recombinant proteins. This is achieved by using the natural tendency of histidine to form a complex with divalent metals around neutral pH. Fusion with the histidine peptides is now one of the most commonly used techniques for affinity purification. Immobilizing the metal ion on a chromatographic resin by chelation allows the separation of the histidine-tagged proteins from most untagged proteins even under denaturing conditions. The binding interaction with the tagged protein is pH dependent. The bound sample can be eluted from the resin by reducing the pH and increasing the ionic strength of the buffer or by including EDTA or imidazole in the buffer. Since target protein can be purified from a large volume of crude lysate in a single step, IMAC purification offers significant time savings over less selective multi-step procedures. Like any other type of chromatography, parameters of purification conditions need to be optimized for the successful practice of any IMAC strategy. These parameters include the choice of metal ion and resin type, the amount of resin to be used, and the condition of binding and elution buffers.

AcroPrep

96 Filter Plate
Increasing demand of protein sample preparation in proteomics accentuates the need to develop more effective strategies for high throughput protein purification. Fully automated purification strategies may include a number of steps where lysate clearance, desalting and protein concentration are needed (3, 4, 5). Beyond standard sample preparation steps, selection of the most suitable protein construct for downstream applications requires the screening of a collection of subclones that often have highly variable expression and activity levels. Performing IMAC on a multi-well filter plate platform is a fast and simple way to purify multiple histidine-tagged proteins in parallel. It allows the high throughput screening of different protein expression constructs. In addition, it facilitates the optimization of various purification parameters including the choice of metal ion resin, the sample-to-resin ratio, and the elution condition.

96 Filter Plate

The AcroPrep 96 filter plate was designed to fit Society for Biomolecular Screening (SBS) compatible manual manifolds as well as robotic equipment. Combined with immobilized metal affinity resin, the plate can be used to purify small amounts of recombinant proteins engineered with a polyhistidine tag. It is ideal for developing protocols for high throughput processing of protein purification.

Specific advantages of using AcroPrep 96 filter plates are:

  • Polypropylene construction is chemically resistant and biologically inert
  • Patented sealing process individually seals membranes preventing lateral flow and crosstalk
  • Rigid single-piece construction
  • Conforms to ANSI/SBS x. 2004 standards for automation
  • Proprietary design minimizes solution/sample weeping

Top

Materials and Methods

Vacuum Filtration
AcroPrep 96 filter plates (Pall Life Sciences)
0.2 µm Bio-Inert® membrane (PN 5042)
0.45 µm GHP membrane (PN 5030)

The use of low biomolecule binding membranes minimizes the sample losses due to non-specific
binding to the plates. In this study, both filtration media demonstrate effective bead retention and low non-specific binding of the proteins.

Vacuum manifold (Pall Life Sciences)
The performance of the AcroPrep filter plates is optimized when used on the Pall Life Sciences vacuum manifold (PN 5017). Other ANSI/SBS x. 2004 compatible manifolds can be used with AcroPrep filter plates.

Reagents

  1. Polyhistidine-tagged TEV protease construct and human Brf2 truncation construct are provided courtesy of Dr. Joshua-Tor’s lab in Cold Spring Harbor Laboratories (Cold Spring Harbor, NY)
  2. HiTrap* Chelating Sepharose HP (Amersham Biosciences, Uppsala, Sweden)
  3. Ni-NTA* Superflow (Qiagen, Valencia, CA)
  4. TALON* Metal Affinity Resin (BD Bioscience, Palo Alto, CA)
  5. Buffer for purification under native condition:
    • Washing Buffer: 100 mM NaPi, 300 mM NaCl, 20 mM Imidazole, pH 8.0
    • Elution Buffer: 100 mM NaPi, 300 mM NaCl, 250 mM Imidazole, pH 8.0
  6. Buffer for purification under denaturing condition:
    • Washing Buffer: 100 mM NaPi, 6M Urea, pH 6.3
    • Elution Buffer: 100 mM NaPi, 6M urea, pH 3.0, 4.0 or 5.0

Top

Step-by-step Procedures

Set-up
  1. Connect the vacuum manifold to a liquid trap.
  2. Connect the liquid trap to a vacuum source through a Vacushield™ vent filter (PN 4402).
  3. Place the collection plate and appropriate spacer block in the lower chamber of the Pall Life Sciences vacuum manifold (PN 5017).
  4. Replace the upper chamber of the vacuum manifold.
  5. Place the AcroPrep 96 filter plate on the gasket located on the top chamber of the vacuum manifold. Ensure that the gasket is clean.

Step-by-step Combinatorial Screen

AcroPrep Plate Spinner
Mixing Resins with Samples Inside individual wells In multiple tubes
Incubation/Binding Directly in plates (no weeping) In multiple tubes
Flow-through Collection Single collect using vacuum manifold Multiple collections
Washing Single collect using vacuum manifold Multiple collections
Elution Single collect using vacuum manifold Multiple collections

Sample Incubation

  1. Mix 100 µL of cell lysate with 20 µL of metal ion resin directly in an AcroPrep96 GHP or Bio-Inertfilter plate. For experiments shown in Figures 1, 3, 4, 5, 6, Ni-NTA* (Qiagen) resin was used. TALON* (BD Bioscience) resin was used in Figure 3. HiTrap Chelating Sepharose (Amersham Biosciences) was used in Figure 2 (charging with Metal Ions described below).
  2. Incubate at 4 °C with gentle rocking for 30 min. 
  3. Alternatively, incubation can be performed in microfuge tubes and transferred into wells of the AcroPrep 96 filter plate for filtration.

    Vacuum Filtration

    1. Place the AcroPrep 96 filter plate on the vacuum manifold and apply vacuum at 25.4 cm Hg (15 in. Hg) for 1 min. Collect the flow-through in a 96-well collection plate.
    2. Wash the resin with 200 µL of washing buffer. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Collect the wash in fresh 96-well collection plates. Repeat twice.
    3. Elute with 100 µL of elution buffer unless otherwise specified. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Collect the elution in fresh 96-well collection plates. Repeat twice.

      Charging Chelating Sepharose with Metal Ions For Custom Resin Screens

      1. Pipette 40 µL of 50% HiTrap* Chelating Sepharose (Amersham Biosciences) slurry into each well of the AcroPrep 96 filter plate.
      2. Place the AcroPrep 96 plate on the vacuum manifold and apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min.
      3. Wash with 200 µL distilled water. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Repeat twice.
      4. Mix the resin with 20 µL of the following solution: 0.1M NiCl2, 0.1M CoSO4, 0.1M CuSO4 and 0.1M ZnCl2, respectively. Incubate for 5 min. at room temperature.
      5. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min.
      6. Wash with 200 µL distilled water. Apply vacuum at 25.4 cm Hg (10 in. Hg) for 1 min. Repeat twice.

        Top

        Results and Discussion

        Sample Incubation Directly in the AcroPrep 96 Filter Plate
        Because the AcroPrep 96 filter plates incorporate low protein-binding filter media and are uniquely designed to prevent weeping from wells, it is possible to perform sample incubation directly in the wells of filter plate. To test this procedure, the sample/resin slurries were incubated either in a microfuge tube (Figure 1, left panel) or directly in a well of an AcroPrep 96 filter plate with 0.2 µm Bio-Inert membrane (Figure 1, right panel) for 30 min. at 4 ºC. The incubation of sample/resin slurry directly in the wells of an AcroPrep 96 filter plate with 0.2 µm Bio-Inert membrane resulted in similar binding efficiency as the incubation in the microfuge tubes. The absence of the His-tagged protein in the flow through lanes from both panels indicated the complete binding of the protein to the resin. The recovery of His-tagged protein was similar for both procedures. In addition, by reducing the volume of each elution aliquot from 200 µL (left panel) to 50 µL (right panel), the protein is completely eluted within the first 100 µL (right panel). This result demonstrates that sample can be incubated in the wells of the AcroPrep 96 filter plates, simplifying the handling procedure and saving time for the analyst.

        Figure 1
        Samples Can be Incubated Directly in a Filter Plate

        Samples Can be Incubated Directly in a Filter Plate
        Aliquots of Ni-NTA resin (Qiagen) were mixed with E. coli inclusion body lysate containing a His-tagged TEV protease construct (load) as described in Step-by-step Procedures. The slurry was either incubated in a microfuge tube and then transferred to a filter plate (left panel) or incubated directly in a well of an AcroPrep 96 filter plate with 0.2 µm Bio-Inert membrane (right panel). After washing, the samples were serially eluted with either 3 X 200 µL (left panel) or 3 X 50 µL (right panel) of elution buffer. The load (L), flow through (FT), wash (W1 and W2), and elution (E1, E2, and E3) were analyzed by SDS-PAGE. The incubation of sample/resin slurry directly in the wells of the filter plate gave similar recoveries to those incubated in microfuge tubes, allowing the simplification of sample handling. Based on this observation, the on-plate incubation procedure was used for the subsequent experiments.

        Custom Resin Screens
        The AcroPrep 96 filter plate allows the parallel screening of metal ions for the purification of a given protein. To select the suitable metal ion, chelating resins can be manually charged with different divalent metal ions. The charged resins are subsequently used in small-scale purification trials to determine the efficiency of the resin. Figure 2 depicts an example of such application. Aliquots of Chelating Sepharose (Amersham Biosciences) resin were charged with Ni2+, Co2+, Cu2+, and Zn2+, respectively, in the individual wells of an AcroPrep 96 filter plate with 0.45 µm GHP membrane. The charged resin was then mixed with E. coli cell lysate (L) containing a His-tagged human Brf2 truncation construct (courtesy of Dr. L. Joshua-Tor of CSHL). As shown in Figure 2, we determined that both Ni2+ and Co2+ charged resins performed well in purifying the tagged protein while Cu2+ and Zn2+ charged resins should not be used. Based on the result using AcroPrep 96 filter plates, we have been able to successfully select the metal ions to be used in purifying the target protein.

        Figure 2
        Screening Manually Charged Resins

        Screening Manually Charged Resins
        The use of an AcroPrep 96 filter plate can help in the selection of the metal ions for successful IMAC purification. Aliquots of Chelating Sepharose (Amersham Biosciences) resin were charged with Ni2+, Co2+, Cu2+, and Zn2+, respectively, in the individual wells of an AcroPrep 96 filter plate with 0.45 µm GHP membrane. The charged resin was then mixed with E. coli cell lysate (L) containing His-tagged human Brf2 truncation construct. The purification was a performed under native condition as described in Step-by-step Procedure. The flow through (FT), wash (W1 and W2), and elution (E1, E2, and E3) were analyzed with SDS PAGE.

        Screening Commercially Available Resins
        The use of an AcroPrep 96 filter plate allows the parallel validation of different commercial resins for the purification of the protein of interest. Reliable purification of human Brf2 truncation construct from the commercially charged resins confirms the ion selection made in Figure 2. We tested two commercial resins; one pre-charged by Ni2+, the other pre-charged by Co2+. Both resins performed well for the protein tested. Because of the popularity of His-tagged recombinant protein, multiple choices of pre-charged IMAC resin are now commercially available for researchers. By using an AcroPrep 96 filter plate, researchers can conveniently screen various resins from different sources and select a pre-charged resin for subsequent purification procedures.

        Figure 3
        Use of Pre-charged Resins

        Use of Pre-charged Resins
        Validation of multiple commercial metal-chelating resins can be easily performed using AcroPrep 96 filter plates. The same lysate samples in Figure 2 were incubated in the wells of an AcroPrep 96 plate with GHP membrane with either Ni-NTA* resin (Qiagen) or TALON* resin (BD Bioscience), respectively. The load (L), flow through (FT), wash (W), and elution (E1, E2, and E3) were analyzed by SDS-PAGE.

        Optimizing IMAC Elution Conditions
        The use of a multi-well filter plate facilitates the side-by-side testing of different elution conditions for protein(s) of interest. This is particularly helpful when a single-step elution condition is to be used instead of an elution gradient. In an AcroPrep 96 filter plate with 0.45 µm GHP membrane (PN 5030), we tested three elution conditions in parallel for the purification of the His-tagged TEV protease. As shown in Figure 4, with all things being equal, the best recovery of the protein under denaturing condition was obtained with elution at pH 3. This result demonstrated that the AcroPrep 96 filter plate could be used to optimize the elution conditions for IMAC purification procedures. 

        Figure 4
        Optimizing Elution

        Optimizing Elution
        Pall Life Sciences AcroPrep 96 filter plate facilitates the optimization of elution conditions for IMAC purification. Aliquots of Ni-NTA* resin (Qiagen) were mixed with E. coli inclusion body lysate (load) containing a His-tagged TEV protease construct. The purification was performed as described in Step-by-step Procedures. After washing, the samples were eluted using three elution buffers with different pH as indicated.

        Optimizing Resin and Sample Loads
        The AcroPrep 96 filter plate with 0.45 µm GHP membrane (PN 5030) was used to determine the loading capacity of the selected IMAC resin prior to large-scale sample purification. In a single experiment, researchers can perform multiple titrations of resin volume and sample load (Figure 5). For a given resin volume of 20 µL, increasing amount of the sample was loaded (upper panel). The resin was saturated at 100 µL sample load as indicated by the appearance of target protein in the flow through (arrows) when larger volume is loaded. Meanwhile, for a given sample volume of 200 µL, increasing amount of resin was used for the purification (lower panel).

        Small amount of resin (40 µL) was sufficient for the complete binding of the His-tagged protein as indicated by the disappearance of target protein in the flow through (arrows) as well as the amount of eluted protein reaching plateau. In both experiments, we have found optimum resin-to-sample ratio was 1:5 (volume). This information was useful in calculating the amount of resin to use in the large-scale purification, minimizing the cost of the sample preparation.

        Figure 5
        Titration of Sample Load versus Resin Volume

        Titration of Sample Load versus Resin Volume
        AcroPrep 96 filter plates can help optimize the amount of resin to be used in an IMAC application. Aliquots of Ni-NTA* resin (20 µL, Qiagen) were mixed with different volumes (5, 10, 20, and 40 µL) of E. coli inclusion body lysate (L) containing a His-tagged TEV protease construct (top panel). In addition, four different volumes of Ni-NTA resin (5, 10, 20, and 40 µL, Qiagen) were mixed with 200 µL of the same lysate (bottom panel). The flow through (FT) and elution were analyzed by SDS PAGE gel.

        Well-to-well Reliability using Multi-well Minicolumns
        High throughput sample processing in proteomic research requires the protein recovery be consistent from well to well in the filter plates. As shown in Figure 6, the elution from 96 identical samples processed by minicolumns in a single AcroPrep filter plate with 0.45 µm GHP membrane (PN 5030) was consistent from well to well as judged by the intensity of protein bands in SDS PAGE gels. In addition, the protein concentration of each eluted sample was quantified by BCA assay, giving a CV of 9.3%. This result indicated superb well-to-well reliability of the AcroPrep 96 filter plate in processing multiple samples.

        Figure 6
        Well-to-well Reproducibility


        Ninety-six 20 µL aliquots of Ni-NTA* resin (Qiagen) were mixed with E. coli inclusion body lysate containing a His-tagged TEV protease construct. The purification was performed as described in Step-by-step Procedures. The eluted samples from each well were analyzed using SDS PAGE. The protein concentration of the elution from separate wells was measured by BCA assay. The coefficient of variation was 9.3%, indicating excellent well-to-well consistency.

        Top

        Conclusions

        IMAC purification is widely used for sample preparations in current proteomic studies. We have demonstrated in this report the use of multi-well filter plates to perform high throughput IMAC. The low-protein binding and no-weeping properties of the AcroPrep 96 filter plate are ideal for convenient incubation of the sample directly in wells. The biomolecule-friendly AcroPrep 96 filter plate allows the researcher to rapidly and reliably screen numerous samples under a variety of conditions to determine protein purification parameters. A single filter plate can be matrixed to:
        • Screen for metal ions for both custom and pre-charged resins
        • Optimize elution conditions
        • Optimize resin-to-load ratio

        In addition, Pall Life Sciences’ AcroPrep 96 filter plate shows consistent well-to-well performance, giving protein biochemists an edge in the development of protein purification protocols.


        Top

        References

        1. Porath J., Carlsson J., Olsson I., and Belfrage G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598-599.
        2. Hochuli E., Bannwarth W., Dobeli H., Gentz R., and Stuber D. (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnology 6: 1321-1325.
        3. Desalting/Buffer Exchange for Biomolecules Using AcroPrep 96 Ultrafiltration Filter Plates, Pall Life Sciences PN33309.
        4. Lysate Clearance for Prokaryotic DNA Isolation Using the AcroPrep 96 Filter Plate, Pall Life Sciences PN33308.
        5. Automated Purification of Combinatorial Libraries Using AcroPrep 96 Filter Plate with GHP Membrane, Pall Life Sciences PN33245.

        Top