High-throughput Screening Using GST MultiTrap FF and GST MultiTrap 4B 96-well Filter Plates

Extracted from GST Gene Fusion System, GE Healthcare, 2014

GST MultiTrap FF and GST MultiTrap 4B (Fig 3.4) are prepacked, disposable 96-well filter plates for reproducible, high-throughput screening of GST-tagged proteins. The plates are filled with a defined amount of affinity medium, Glutathione Sepharose 4 Fast Flow (4% highly cross-linked agarose beads) or Glutathione Sepharose 4B (4% agarose beads). Each well contains 500 µl of 10% slurry of the medium in 20% ethanol as storage solution. Typical applications include expression screening of different constructs, screening for solubility of proteins, and optimization of the conditions for small-scale parallel purifications. These filter plates simplify the purification screening and enrichment of up to 0.5 mg of GST-tagged proteins/well. Note that binding depends on the flow and may vary between proteins. Incubation of the sample with medium is necessary, and optimization for optimal binding of the GST-tagged protein is recommended. It is also possible to apply up to 600 µl of unclarified lysate, after thorough cell disruption, directly to each of the wells without precentrifugation and/or filtration of the sample. It is recommended to extend the duration of mechanical/chemical lysis if the sample is too viscous after lysis; alternatively, include nucleases to disrupt nucleic acids. The GST-tagged proteins are eluted under mild, nondenaturing conditions to preserve protein structure and function.

The 96-well filter plates with 800 µl volume capacity per well are made of polypropylene and polyethylene. Characteristics of GST MultiTrap FF and GST MultiTrap 4B are listed in Appendix 5 (Characteristics of Glutathione Sepharose and HiTrap Benzamidine FF (High Sub) Media and Columns).

Prepacked GST MultiTrap FF and GST MultiTrap 4B plates give high reproducibility well-to-well and plate-to-plate, allowing parallel screening of chromatographic conditions. The repeatability of yield and purity of eluted protein is also high. The plates can be used in automated workflows using robotic systems, or can be operated using centrifugation or by vacuum pressure. The purification protocol included with the plates can easily be scaled for use with the different prepacked formats: GST GraviTrap, GSTrap FF, and GSTrap 4B (1 ml and 5 ml columns) and GSTPrep FF 16/10 (20 ml column) as discussed later in this chapter.

Fig 3.4. GST MultiTrap FF and GST MultiTrap 4B 96-well filter plates.

Fig 3.4. GST MultiTrap FF and GST MultiTrap 4B 96-well filter plates.

Sample preparation

Adjust the sample to the binding buffer conditions by diluting it with binding buffer or by buffer exchange.

After thorough cell disruption, it is possible to apply unclarified lysate directly to the wells without precentrifugation or filtration of the sample. The unclarified lysate should be used directly after preparation, as the lysate may precipitate. The unclarified lysate can also be frozen until use but needs to be lysed again before starting the procedure.

Lysis with commercial kits may give large cell debris particles that may interfere with drainage of the wells during purification. This problem can be solved by centrifugation or filtration of the sample before applying to the wells. The binding properties of the target protein can be improved by performing a buffer exchange using a PD MultiTrap G-25 96-well filter plate.

Before starting the procedure, refer to page 32 for general considerations for purification of GST-tagged proteins.

Reagents required

Use high-purity water and chemicals, and pass all buffers through 0.45 µm filters before use.

Binding buffer: PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.3
Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

1 to 20 mM DTT can be included in the binding and elution buffers to reduce the risk of oxidation of free -SH groups of GST. Oxidation may cause aggregation of the tagged target protein, resulting in lower yield of GST-tagged protein.

Centrifugation Procedure
  1. Hold the 96-well filter plate horizontally over a sink and carefully peel off the bottom seal.

  2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal.

  3. Reposition the filter plate upright, and place it on a bench, then peel off the top seal.

Note: If the medium has dried out in one or several wells, add buffer to rehydrate. The performance of the medium is not affected.

  1. Place the filter plate on top of a collection plate.

Note: Remember to change or empty the collection plate as necessary during the following steps.

  1. Centrifuge the filter plates for 2 min at 500 × g, to remove the storage solution from the medium.

  2. Add 500 µl of deionized water per well and centrifuge for 2 min at 500 × g.

  3. Add 500 µl of binding buffer per well, and mix briefly to equilibrate the medium. Centrifuge for 2 min at 500 × g. Repeat entire step once.

Do not apply more than 700 × g for centrifugation.

  1. Apply unclarified or clarified lysate (maximum 600 µl per well) to the wells and incubate for 3 min.

Note: For increasing the protein yield, gently shake the filter plate for an effective mixing and/or increase the incubation time.

  1. Centrifuge the plate at 100 × g for 4 min or until all the wells are empty. Discard the flowthrough.

  2. Add 500 µl of binding buffer per well to wash out any unbound sample. Centrifuge at 500 × g for 2 min. Repeat once or until all unbound sample is removed.

Note: Removal of unbound material can be monitored by measuring A280. An A280 value  < 0.1 indicates effective removal of the unbound sample.

  1. Add 200 µl of elution buffer per well and mix for 1 min.

Note: For higher protein concentration in the eluted sample, the elution volume can be changed between 50 and 100 µl. Smaller volume may give uncertain absorbance values.

  1. Change the collection plate and centrifuge at 500 × g for 2 min to collect the eluted protein. Repeat twice or until all of the target protein has been eluted, as monitored by A280 measurement.

Note: The collection plate can be changed and collected separately between each elution step to avoid unnecessary dilution of the target protein.

Vacuum Procedure

If problems with foaming, reproducibility, or bubbles in the collection plate occur using vacuum, the centrifugation procedure should be considered.

The distance between the bottom of the filter plate and the top of the collection plate in the vacuum manifold should be about 5 mm to avoid cross-contamination in the collection plate. Place an appropriate spacer block into the lower chamber of the vacuum manifold to reduce the distance between the plates.

If a robotic system is used for purification, the vacuum must be adjusted according to methods applicable to the system.

Do not apply a pressure higher than -0.5 bar during vacuum operation.

  1. Hold the 96-well filter plate horizontally over a sink and carefully peel off the bottom seal.

  2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal.

  3. Reposition the filter plate upright, and place it on a bench, then peel off the top seal.

Note: If the medium has dried out in one or several wells, add buffer to rehydrate. The performance of the medium is not affected.

  1. Place the filter plate on top of a collection plate.

Note: Remember to change or empty the collection plate as necessary during the following steps.

  1. Set the vacuum to -0.15 bar. Place the filter plate and collection plate on the vacuum manifold to remove the storage solution from the wells. Turn off the vacuum as soon as all the solution is removed, to avoid cross-contamination in the collection plate.

  2. Add 500 µl of deionized water to each well and apply vacuum. Maintain vacuum until all liquid passes through the wells.

  3. Add 500 µl of binding buffer to each well to equilibrate the medium. Apply vacuum as in step 5. Repeat once. The filter plate is now ready to use.

  4. Apply unclarified or clarified lysate (maximum 600 µl per well) to the wells of the filter plate and incubate for 3 min.

Note: For increasing the protein yield, gently shake the plate and/or increase the incubation time.

  1. Vacuum (−0.15 bar) until all liquid passes through the filter plate and until all the wells are empty. Slowly increase the vacuum to −0.30 bar and turn off the vacuum after approximately 5 s. Discard the flowthrough.

Increasing the vacuum too quickly can result in foaming under the filter plate with subsequent cross-well contamination as the consequence.

  1. Add 500 µl of binding buffer per well to wash out any unbound sample. Apply vacuum of -0.15 bar as in step 9. Repeat once or until all unbound sample is removed.

Note: Removal of unbound material can be monitored by measuring A280. An A280 value < 0.1 indicates effective removal of the unbound sample.

  1. Add 200 µl of elution buffer per well and mix for 1 min.

Note: The volume of elution buffer can be varied (50 µl to 100 µl per well), depending on the required concentration of target protein. Smaller volumes may give uncertain absorbance values when measuring A280.

  1. Change the collection plate and apply vacuum of −0.15 bar to collect the eluted protein. Repeat twice or until all of the target protein has been eluted, as monitored by measuring A280.

Note: The collection plate can be changed and collected separately between each elution step to avoid unnecessary dilution of the target protein.

Application example

Screening and purification of GST-hippocalcin using GST MultiTrap FF

In this example, the conditions of the binding buffer were optimized for purification of GST-hippocalcin using GST MultiTrap FF. A buffer-screening study to determine optimal buffer conditions for the purification was designed including pH, sodium chloride, glycerol, DTT, and glutathione amount. A comparison between sonication and use of a commercial cell lysis kit was also performed. Factorial design (design-of-experiments) and statistical analysis were performed using MODDE™ software. The different buffer conditions and sample preparation methods were randomly applied and tested on the filter plate.

The screening results showed that the optimal buffer conditions for purifying GST-hippocalcin with the highest yield and purity were: 10 to 20 mM sodium phosphate, 140 to 400 mM NaCl, pH 6.2 to 7.0 (data not shown). Moreover, the results showed that either the commercial cell lysis kit or sonication can be used to lyse E. coli without any significant changes in the purification result (Fig 3.5).

The presence of glutathione in the sample and binding buffer (also used as wash buffer) decreased the yield of purified GST-hippocalcin significantly, while the different types of buffer had no effect. Low pH improved the yield whereas high pH (8.0) affected the yield negatively. No significant effect on purity (Fig 3.5) was seen with changing the pH. Additives such as DTT, glycerol, and NaCl did not significantly affect the yield or purity of this particular protein.

Sample preparation

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Fig 3.5. Coomassie-stained SDS-polyacrylamide gel (ExcelGel™ 8–18%) of collected eluted GST-hippocalcin fractions from some of the GST MultiTrap FF filter plate wells.

Materials

     
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