Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

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Purification of recombinant protein from bacteria is a well-established method. However, several problems may occur when using these techniques. Having methods to extract protein from tissue is for sure appealing.

The main advantage with this method is that one may extract the protein in its physiological form including all mutual effects that may impact its folding and catalytic activity. This approach may be adapted to other proteins of interest and modified for other tissue. Purified protein from tissue may support the development of high-quality antibodies and pharmacological inhibitors.

To begin, prepare protein lysis buffer by mixing 250 milliliters of PBS with 50 millimolar sodium fluoride, one millimolar phenylmethylsulfonyl fluoride, two micrograms per milliliter aprotinin, and one millimolar activated orthovanadate. Filter the solution using a 0.22 micrometer syringe filter unit. Prepare tubes with two milliliters of lysis buffer per gram of tissue and place them on ice.

To prepare the tissue sample, dissect the tissue of about 100 milligrams each on a pre-cleaned glass plate placed on ice in a polystyrene foam box. Transfer the tissue pieces into the prepared tubes for subsequent lysis. To prepare a saturated ammonium sulfate solution, heat 500 milliliters of double distilled water to 70 degrees Celsius.

While stirring, gradually add ammonium sulfate powder until no more ammonium sulfate is dissolved. Cool this saturated solution to room temperature and store it at four degrees Celsius overnight. To homogenize the swine kidney tissues, sonicate the suspension preferably by an ultrasonic probe while keeping the sample on ice.

In the case of mouse liver tissues, use an electric homogenizer and wash it regularly in PBS to prevent clogging while keeping the samples on ice. Take 20 microliters out of the samples and check under the microscope whether the cells of the homogenized tissue are properly destroyed. Otherwise, repeat the homogenization.

Centrifuge the tubes at 10, 000 times G for 30 minutes at four degrees Celsius. Collect the supernatant in a fresh tube and place it on ice. Subsequently filter the supernatant using 0.45 micrometer and 0.22 micrometer syringe filter units.

Aliquot the supernatant into 10 milliliter batches and freeze them at minus 20 degrees Celsius for short-term storage or at minus 80 degrees Celsius for longer storage. Prepare a 50 microliter sample for SDS-PAGE or Western blot analysis by adding 10 microliters of five times the SDS sample buffer to 40 microliters of the supernatant and then boiling at 95 degrees Celsius for 10 minutes. Prepare a discontinuous 12.5%polyacrylamide SDS-PAGE gel according to the manufacturer's instructions.

To perform SDS-PAGE analysis, load the wells of the gel subsequently with a protein marker ladder, five nanograms of human FAHD1 recombinant protein as a positive control, 20 microliters of the sample to be analyzed, and the remaining wells with 20 microliters of prepared SDS-PAGE sample buffer. Run the SDS-PAGE gels at 125 volts using the SDS running buffer. After SDS-PAGE is complete, perform a Western blot analysis, probe the membranes using the available antibody raised against FAHD1.

After preparing supernatant aliquots as described previously, either process after thawing or process directly after protein extraction without freezing the sample. Filter the sample using a 0.22 micrometer filter unit to exclude possible precipitates after thawing. Prepare six 1.5 milliliter tubes on ice, then transfer 250 microliters of sample into each tube.

Prepare a dilution series of ammonium sulfate in the prepared tubes and make up the final volume to 1, 000 microliter with protein lysis buffer. Incubate the samples at four degrees Celsius overnight on a tube rotator. Using a tabletop centrifuge, centrifuge at 10, 000 times G for 30 minutes at four degrees Celsius and carefully transfer all of the supernatants into separate tubes.

Air dry the resulting pellets and resuspend each of them in 1, 000 microliter of double distilled water. For each pair of resuspended pellet and supernatant from the previous step, mix 40 microliters with 10 microliters of five times the SDS sample buffer and boil at 95 degrees Celsius with open lids until most of the liquid has vaporized. Then resuspend the pellet in a mixture of 50%dimethyl sulfoxide in double distilled water.

Perform SDS-PAGE by loading the samples derived from the resuspended pellets and supernatant in pairs, followed by a Western blot analysis. However, run the gels at 80 volts for three hours. Set up the FPLC system with the anionic or cationic exchange column.

Wash the column with five column volumes of 20%ethanol in double distilled water, followed by five column volumes of double distilled water. Apply the sample onto the column either by injection or by using a sample pump and collect the flow-through. Wash the column with one column volume of the low salt buffer.

Set up a linear gradient elution from 100%low salt buffer to 100%high salt buffer within three column volumes. Continuously collect one milliliter fractions. After the gradient has finished, continue to run with a high salt buffer until no more protein-associated peaks are detected in the chromatogram over the range of one column volume.

Apply one milliliter of 25%SDS dissolved in 0.5 molar sodium hydroxide and double distilled water to clean the column. Consecutively wash the column with three column volumes of double distilled water and three column volumes of 20%ethanol in double distilled water. Collect the SDS-PAGE samples of all peak fractions and the flow-through.

Snap freeze the collected fractions in liquid nitrogen and store them at minus 80 degrees Celsius. After probing them via Western blot, thaw and pool the fractions containing the proteins of interest and discard the others. To perform the purification step, reduce the volume of the protein solution down to two milliliters using ultra centrifugation filter units.

Sequentially filter the solution with 0.45 micrometer and 0.22 micrometer syringe filter units to remove any microprecipitation. Equilibrate the size exclusion chromatography column with one column volume of running buffer containing one millimolar dithiothreitol. Load the sample onto the column and run the chromatography unit until all the proteins are eluted as described previously.

Prepare 50 microliter samples for SDS-PAGE and Western blot analysis described previously. Consecutively wash the column with one column volume of double distilled water and one column volume of 20%ethanol in double distilled water. After performing Western blot analysis, pool all FAHD1 containing fractions.

Reduce the volume of the protein solution down to two milliliters using ultra centrifugation filter units. Western blot screening shows the presence of swine FAHD1 in the supernatant with a band at 25 kilodaltons while the tagged positive control runs at 34 kilodaltons. The swine FAHD1 from lysate was purified by anionic exchange chromatography and the binding proteins were removed by elution.

Fractions containing swine FAHD1 were identified by Western blot analysis, pooled and concentrated. Protein samples were purified by SEC and identified by Western blot analysis. The yield of swine FAHD1 protein was about one milligram with an 80%level of purity.

Western blot analysis detected the mouse FAHD1 protein in the flow-through along with other proteins. The flow-through was applied for size exclusion chromatography. Fractions containing FAHD1 were pooled, concentrated, and applied to SDS-PAGE analysis.

However, the yield of protein was not very high. The specific enzymatic activity of the swine FAHD1 increases with the purity level upon increasing the relative amount of FAHD1 per total protein. Higher concentrations of ammonium sulfate cause the smile effect, which can be resolved at lower voltage.

Further protein was found in the lysate and supernatant at 15%concentration. However, at higher concentrations, samples may precipitate and cannot be detected. The protocol presented here requires that the protein is solvable.

Optimal conditions for precipitation and pH or size adjustment for chromatography need to be tested and defined. The study of enzymatic function, the investigation of protein structure and the development of antibody are greatly supported when having protein extracted from tissue rather than extracted from bacteria.

Journal (Biochemistry)

Journal (Biochemistry)

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

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