Performing a Small-Scale Expression Screening of Histidine-Tagged Membrane Proteins from E. coli Lysates

Extracted from Purifying Challenging Proteins - Principles and Methods, GE Healthcare, 2007

Cell lysis and solubilization

Buffer preparation

Lysis buffer: 20 mM sodium phosphate, 100 mM NaCl, 2 mM MgCl2, 20 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 5 u/mL benzonase, 1 mg/mL lysozyme, EDTA-free protease inhibitor cocktail, (concentration according to manufacturer’s recommendation), 1-2% of a selection of detergents, pH 7.4

Procedure

1. 1. Harvest cells from the culture by centrifugation at 7000 to 8000 × g for 10 min or at 1000 to 1500 × g for 30 min at 4 °C.
2. 2. Discard the supernatant. Place the bacterial pellet on ice.
3. 3. Suspend the bacterial pellet by adding 5 to 10 mL of lysis buffer for each gram of wet cells. To prevent the binding of host cell proteins with exposed histidines, it is essential to include imidazole at a low concentration in the sample and binding buffer.
4. 4. Leave for 2 h with mild agitation at room temperature or 4 °C, depending on the sensitivity of the target protein.
5. 5. Measure and adjust pH if needed.

Expression screening procedure

Materials

• His MultiTrap HP or His MultiTrap FF
• Centrifuge that handles 96-well plates

Buffer preparation

Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, 0.5 mM TCEP, 1 to 2% detergent, pH 7.4. (The optimal imidazole concentration is protein dependent; 20 to 40 mM is suitable for many proteins.)

Wash buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, 0.5 mM TCEP, 0.03% dodecyl maltoside (DDM), 1 to 2% detergent, pH 7.4,

Elution buffer: 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, 0.5 mM TCEP, 0.03% DDM, 1 to 2% detergent, pH 7.4

To increase the purity, use as high a concentration of imidazole as possible in the sample and binding buffers without losing binding capacity.

Preparing the filter plate

1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over a sink to accommodate any leakage of storage solution when removing the bottom seal.
2. Hold the filter plate upside down and gently shake it to dislodge any medium adhering to the top seal. Return the filter plate to an upright position.
3. Place the filter plate against the bench surface and peel off the top seal.
4. Position the filter plate on top of a collection plate. Note: Remember to change or empty the collection plate as necessary during the following steps.
5. Centrifuge the filter plate for 2 min at 200 × g to remove the ethanol storage solution from the medium.
6. Add 500 µL of deionized water to each well. Centrifuge the plate for 2 min at 200 × g.
7. Add 500 µL of binding buffer to each well to equilibrate the medium. Centrifuge for 2 min at 200 × g. Repeat once. The filter plate is now ready for use.
   Blank run: Reducing agents may be used in sample and buffers. If this is the case, replace step 7 with the following steps:
8. Add 500 µL of elution buffer/well. No reducing agent should be used in the elution buffer during this blank run. Centrifuge the plate for 2 min at 200 × g.
9. Add 500 µL of binding buffer including reducing agent to each well to equilibrate the medium. Centrifuge for 2 min at 200 × g. Repeat once. The filter plate is now ready for use with reducing agent. Do not store His MultiTrap plates with buffers containing reducing agents.

Centrifugation procedure

Check that all wells are drained after centrifugation. If not, then increase the centrifugation force slightly.

Do not apply a force of more than 700 × g during centrifugation.

1. Apply 100 µL of lysate to each well of the filter plate and incubate for 3 min.
   Note: If the yield of protein is very low, increase the incubation time and/or gently agitate the filter plate to mix. The lysate volume can also be increased, and several aliquots of lysate can be added successively to each well.
2. Centrifuge the plate at 100 × g for 4 min or until all the wells are empty. Discard the flowthrough.
3. Add 50 µL of binding buffer per well. Centrifuge at 200 × g for 2 min.
4. Add 200 µL of wash buffer per well. Centrifuge at 200 × g for 2 min. Repeat twice.
5. Add 50 µL of elution buffer per well and mix for 1 min.
   Note: The volume of elution buffer can be varied (50 to 600 µL per well), depending on the concentration of target protein required.
6. Change the collection plate and centrifuge at 200 × g for 2 min to collect the eluted protein. Repeat twice or until all the target protein has been eluted (A₂₈₀ should be < 0.1, indicating that all protein has been eluted).
   Note: If necessary, change the collection plate between each elution to prevent unnecessary dilution of the target protein.

The following detergents have been used with this protocol: 1% FOS-Choline™ 12, 1% undecyl maltoside, 1% dodecyl maltoside, 1% Cymal™-5, 1% Cymal-6, 2% octyl glucoside, 1% Triton™ X-100, 1% lauryl dimethylamine oxide (LDAO).

To optimize the protocol, vary the concentration of imidazole in the sample and in the binding and wash buffers. A common variation range with His MultiTrap plates is 20 to 40 mM imidazole. If binding of the target protein is too low with these concentrations, try 5 to 20 mM imidazole. In general, too low of an imidazole concentration in the binding and wash buffers can cause adsorption of unwanted host proteins (and hence a lower purity). Too high of an imidazole concentration can lead to a reduced yield of the target protein.

Analysis

Samples can by analyzed by SDS-PAGE with Coomassie™ Blue staining (Performing a purity and homogeneity check), or by dot-blot analysis on nitrocellulose membrane. Histidine-tagged proteins can be detected using Anti-His Antibody.

Cell harvest

Methods for cell harvest are host dependent and the same protocols are used for the recovery of membrane proteins as for intracellular water-soluble proteins. Cell harvest of suspension cultures is done by low speed centrifugation.

Cell harvest of E. coli cultures

Buffer preparation

PBS: 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4

Centrifugation procedure

1. Centrifuge at 6000 to 9000 × g at 4 °C for 15 min to collect the cells. Discard the supernatant.Resuspend the cells in 500 mL ice-cold PBS.

1. Centrifuge at 6000 to 9000 × g at 4 °C for 15 min. Discard the supernatant. Resuspend the cell pellet in 10 mL ice-cold PBS, or another volume as required.
2. The resuspended cell pellet can be stored at –80 °C.