Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Our protocol describes methods for efficient expression and purification of FAH domain containing proteins but also proteins in general. FHD protein-1 plays a regulatory role in the TCA cycle and the energy metabolism of mitochondria. We were able to associate FHD-1 downregulation to cellular senescence and information on enzyme activity and protein structure are generally required in order to understand molecular mechanisms behind observed phenotypes.

The main advantage of expressing proteins via IPTG inducible vector systems is that all methods are relatively cheap and easy to establish in any laboratory. By using poly-histidine type proteins in combination with nickel NTA agarose resins, the immense selectivity of affinity chromatography may be employed at low cost. For most purposes, protein obtained at this quality may already suffice.

FPLC on the other hands, is a well established and general method with several advantages compared to other methods. First, insert five to 10 nanograms of plasmid into 100 microliters of competent BL21-DE3 pLysS E.coli bacteria on ice. Slightly tap the tube in order to mix the contents.

Keep the bacteria on ice for 30 minutes, gently tapping the tube every few minutes. Heat a thermo shaker to 42 degrees celsius. Place the tube containing the bacteria in the apparatus and heat for 90 seconds.

Then, place the tube on ice immediately. After five to 10 minutes on ice, add 600 microliters of NZCYM medium and place the tube into a bacteria incubator. Shake the tube at medium speed oriented along the shaking direction at 37 degrees celsius for one hour.

Following incubation, plate 200 microliters of the bacterial culture on a 10 centimeter LB agar plate containing the selection antibiotics of choice. Culture the bacteria on the LB agar plate in the bacteria incubator at 37 degrees celsius overnight. After successful colony formation, pick one single colony and disperse it in five milliliters of NZCYM with the selected antibiotics.

Culture in the bacteria incubator at 37 degrees celsius overnight. After successful bacterial growth, amplify the bacteria in 250 milliliter, 500 milliliter or one liter batches of medium depending on the demand of protein quantity. Then, culture the bacteria, in the bacteria incubator at 37 degrees celsius for two to three hours.

Following incubation, draw a sample for photometric analysis. If the optical density at 600 nanometers has reached 0.4, apply 200 micromolar up to one millimolar of isopropyl-beta-D-thiogalactopyranosid. After three to five more hours in the bacteria incubator at 37 degrees celsius protein expression is exhausted.

Harvest the bacterial pellet via centrifugation at 5, 000 times G for five minutes. Then, discard the supernatant and freeze the pellet at minus 20 degrees celsius for brief storage. For each 250 milliliters of original bacterial suspension, apply five milliliters of the selected buffer to the bacterial pellet.

Add 10 microliters of beta-mercaptoethanol per five milliliters of applied buffer. Use a 10 milliliter Pasteur pipet to mechanically force the pellet into suspension by scratching and pipetting. Transfer all of the suspension to a 50 milliliter tube.

Then sonicate the suspension. Next, centrifuge for 30 minutes at high speed at four degrees celsius. Filter the supernatant consecutively with filter units on ice.

Prepare an empty plastic or glass column by attaching it to a stable retainer and washing with nickel NTA running buffer. For each 10 milliliters of protein suspension apply 500 microliters of nickel NTA agarose slurry to the column. Fill the column completely with nickel NTA running buffer ensuring not to disrupt the agarose resin and allow the buffer to run through by gravity.

Next, apply the protein suspension to the column and allow the sample to run through by gravity. After the sample has passed through, fill the column with nickel NTA running buffer. Place a UV transparent cuvette below the column and collect one milliliter of nickel NTA elution buffer.

Check the optical density of the sample at 280 nanometers versus a blank sample. Optimally, the sample displays an optical density of greater than 2.5. Since FAHD proteins and nickel NTA elution buffer will precipitate upon freezing and thawing, dialyze the protein against a different buffer overnight on ice using one microliter of DTT per 100 milliliters of dialysis buffer.

Set up an FPLC system and wash the column with five column volumes of 20%ethanol followed by five column volumes of water. After equilibrating the column, apply the dialyzed sample to the column and collect the flow through. Set up a gradient elution.

After the gradient has finished, run with high salt buffer until no more peaks are detected over the range of one column volume. Start up a micro-plate reader and equilibrate for 30 minutes at 25 degree celsius. Prepare one milliliter of a 20 millimolar solution of a substrate to be tested in enzyme assay buffer.

According to the pipetting scheme in the text protocol, prepare the enzyme blank and sample wells by pipetting 90 microliters of enzyme assay buffer into the wells with five microliters of enzyme solution. Then prepare the substrate blank and sample wells by pipetting 95 microliters of enzyme assay buffer into the wells. Right before measuring, apply five microliters of enzyme assay buffer to the six blank wells.

Apply five microliters of the 20 millimolar substrate solution to the sample wells. Use a multi-channel pipet at 50 microliter setting to gently mix the wells. Then, insert the plate into the micro-plate reader and measure each well at 255 nanometers.

Finally, perform the analysis in a spreadsheet. Two examples LB agar plates after optimal and non-optimal transformation are depicted here. Too many bacterial colonies indicate either that two many bacteria were plated or that the antibiotics may be expired.

Too few bacterial colonies may indicate that either not enough plasmid was used for the transformation or that too much antibiotics were used to select the bacteria. The bacterial pellet containing the expressed protein in milligram quantities is harvested and expression is verified via SDS-page. Some problems may occur during this otherwise simple process, including the proteins forming inclusion bodies or the protein not being expressed.

If a His-tag is used to tag the protein, affinity chromatography with nickel NTA agarose is an easy and cheap capture method eliminating the majority of contaminations. If no tag is used, a combination of ammonium sulfate precipitation and a consecutive hydrophobic exchange chromatography may also separate the protein from the majority of other proteins. The protein is further separated from leftover contaminations by ion exchange chromatography followed by size exclusion chromatography to obtain a sufficiently pure protein.

None of the described methods is difficult in particular. But all methods require training. First attempts may fail but the experiments will succeed after some additional adaptations.

All methods presented here are general methods of protein purification. Although specialized for FAHD proteins, the protocol may be adapted to any protein one would like to purify. Recombinant human FAHD-1 has been obtained by the described methods and high resolution x-ray structures contributed massively to our understanding of FAHD-1 by functionality acting as both, decarboxylase and hydrolase.

Bacteria used in this work are safety strains and not hazardous. General safety guides apply to all chemicals and FPLC columns should be handled with care.