Screening pGEX Recombinants for Tagged Protein Expression

Extracted from GST Gene Fusion System, GE Healthcare, 2014

Sections of this procedure have been adapted with permission from Current Protocols in Molecular Biology, Vol. 2, Supplement 10, Unit 16.7. Copyright © 1990 by Current Protocols.

The following steps may be used prior to large-scale purification to check clones for expression of the desired tagged protein.

Reagents Required

Preparation of the medium

Bulk Glutathione Sepharose 4B prepared to 50% slurry as described below in the procedural steps

PBS:  140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, pH 7.3.

Preparation of lysate

2× YTA medium (2× YT + 100 µg/ml ampicillin): Prepare 2× YT medium by dissolving 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl in 900 ml of distilled water. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 l with distilled water. Sterilize by autoclaving for 20 min. After autoclaving, cool the medium to 50°C, then aseptically add 1 ml of a 100 mg/ml ampicillin stock solution (final concentration 100 µg/ml).
100 mM IPTG:  Dissolve 500 mg of isopropyl-β-D-thiogalactoside (IPTG) in 20 ml of distilled water. Filter-sterilize and store in small aliquots at -20°C.
Elution buffer:  50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Dispense in 1 to 10 ml aliquots and store at -20°C until needed. Avoid more than five freeze/thaw cycles.
Procedure

Preparation of the medium

Glutathione Sepharose 4B is supplied preswollen in 20% ethanol. The medium is used at a final slurry concentration of 50%.

  1. Determine the bed volume of Glutathione Sepharose 4B required.

Although only 10 µl of prepared slurry is needed for each screening analysis, additional slurry should be prepared if it will also be used for larger-scale purification procedures (see Batch purification using Glutathione Sepharose 4B and Batch/column purification using Glutathione Sepharose 4B in Chapter 3).

  1. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the medium.

  2. Use a pipette with a wide-bore tip to remove sufficient slurry for use and transfer the slurry to an appropriate container/tube.

  3. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant.

  4. Wash the Glutathione Sepharose 4B by adding 5 ml of PBS per 1 ml of slurry (=50% slurry). Invert to mix.

Glutathione Sepharose 4B must be thoroughly washed with PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures.

  1. Sediment the medium by centrifuging at 500 × g for 5 min. Carefully decant the supernatant.

  2. Repeat steps 5 and 6 once for a total of two washes. Add PBS to obtain a 50% slurry.

Note: The bed volume is equal to half of the volume of the 50% slurry.

Preparation of lysate

  1. Pick and transfer several colonies of E. coli transformed with the pGEX recombinants into separate tubes containing 2 ml of 2× YTA medium.

For comparison, it is advisable to inoculate a control tube with bacteria transformed with the parental pGEX plasmid.

  1. Grow liquid cultures to an A600 of 0.6 to 0.8 (3 to 5 h) with vigorous agitation at 30°C to 37°C.

Lower temperatures, even as low as 20°C, may be used if inclusion bodies are problematic.

  1. Induce tagged protein expression by adding 2 µl of 100 mM IPTG (final concentration 0.1 mM).

A higher concentration (up to 1 mM IPTG) may be used at this screening stage.

  1. Continue incubation for an additional 1 to 2 h.

  2. Transfer 1.5 ml of the liquid cultures to labeled microcentrifuge tubes.

  3. Centrifuge in a microcentrifuge for 5 s and discard the supernatants.

  4. Resuspend each pellet in 300 µl of ice-cold PBS. Transfer 10 µl of each cell suspension into separate labeled tubes (for later use in SDS-PAGE analysis).

Except where noted, keep all samples and tubes on ice.

  1. Lyse the cells using a sonicator equipped with an appropriate probe or other mechanical methods available. Alternatively, use chemical lysis buffers for protein extraction.

Lysis is complete when the cloudy cell suspension becomes translucent. The frequency and intensity of sonication should be adjusted such that complete lysis occurs in 10 s, without foaming (foaming may denature proteins). Keep on ice.

  1. Crude lysates can be screened for the relative level of expression of GST-tagged proteins using the GST substrate CDNB. See GST Detection Module with CDNB enzymatic assay, Chapter 4.

  2. Centrifuge the lysate in a microcentrifuge for 5 min to remove insoluble material. Save a 10 µl aliquot of the insoluble material for analysis by SDS-PAGE. Transfer the supernatants to fresh tubes.

  3. Add 20 µl of a 50% slurry of Glutathione Sepharose 4B (prepared as described above) to each supernatant and mix gently for 5 min at room temperature.

  4. Add 100 µl of PBS, vortex briefly, and centrifuge for 5 s to sediment the Glutathione Sepharose 4B beads.

  5. Discard the supernatants. Repeat this PBS wash twice for a total of three washes.

  6. Elute the tagged protein by adding 10 µl of elution buffer. Suspend the Glutathione Sepharose 4B beads and incubate at room temperature for 5 min.

Centrifuge in a microcentrifuge for 5 min to sediment the Glutathione Sepharose 4B beads, then transfer the supernatants to fresh tubes for SDS-PAGE analysis.

Transformants expressing the desired tagged protein will be identified by the absence from total cellular proteins of the parental GST and by the presence of a novel, larger tagged protein. Parental pGEX vectors produce a Mr 29 000 GST-tagged protein containing amino acids coded for by the pGEX MCS.

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