The overall goal of this protocol is to overexpress and purify codon-optimized human cis-prenyltransferase under non-denaturing conditions and to assay its enzymatic activity. The procedure is demonstrated with the eukaryotic long-chain cis-prenyltransferase dehydrodolichyl diphosphate synthase, or DHDDS. This method can advance our understanding of isoprenoid synthesis and provide key insights into the pathophysiological mechanism of retinitis pigmentosa related to mutations in DHDDS.
The main advantages of this technique is that it's simple, cost-effective, and time-saving. It also can be generalized for high-quantity production of other cis-prenyltransferase proteins. Begin this procedure with cloning an expression of human DHDDS, as described in the text protocol.
Resuspend the cells in buffer A, one microgram per milliliter of DNase I, and a protease inhibitor mixture. Then, homogenize the resuspended cells using a glass Teflon homogenizer. Disrupt the cells with a Microfluidizer or equivalent at 12, 000 to 15, 000 psi.
Next, centrifuge the cell lysate at 40, 000 times g for 45 minutes at four degrees Celsius. Recover the supernatant containing the soluble fraction. Load the supernatant onto a five to 10 millimeter cobalt-immobilized metal affinity chromatography column with 10 millimolar imidazole.
After loading into a fast protein liquid chromatography machine, perform a thorough washing of the column with buffer A in 10 millimolar imidazole in order to reduce non-specific protein binding. Begin the FPLC program. Elute the overexpressed proteins with buffer A supplemented with 250 millimolar imidazole.
Following elution, remove the imidazole using 53 milliliters of a preparative desalting column equilibrated with buffer A.Then, add hexahistidine-tagged TEV protease to the eluted proteins to remove their hexahistidine-tagged thioredoxin DHDDS fusion. Incubate the mixture at four degrees Celsius overnight. Next, load the cleaved proteins onto a cobalt-immobilized metal affinity chromatography column equilibrated with buffer A supplemented with five millimolar imidazole.
Collect the flowthrough to remove the cleaved hexahistidine-tagged thioredoxin and TEV protease. Concentrate the flowthrough to between three and four milliliters using a 30 kilodalton molecular weight cut off centrifugal filter. Then, load the concentrated protein onto a Superdex 200 size exclusion chromatography column equilibrated with buffer A for final purification.
Place the samples into a 96-well rack in a fraction collector. The protein elutes as a dimer. Select relevant fractions by comparing the elution profile to a column calibration curve.
At this point, assess the purity of the preparation using SDS-PAGE. Finally, determine the protein concentration needed for downstream experimentation and flash freeze aliquots of purified concentrated proteins in liquid nitrogen. Store the aliquots at minus 80 degrees Celsius until use.
To ensure the ability of the protein to endure freeze-thaw cycles, thaw an aliquot of the frozen proteins. Centrifuge the aliquot at 21, 000 times g for 10 minutes to remove insoluble aggregates. Following centrifugation, collect the supernatant.
Next, load the proteins onto a Superdex 200 analytical column pre-equilibrated with TNXB using an ultra performance liquid chromatography system. Monitor the tryptophan fluorescence to detect the protein's elution profile. Be sure to have a valid calibration curve for molecular weight assessment, which is available via the SEC column manufacturers.
To ensure the activity of the purified protein, first mix five micromolar of purified DHDDS with 10 micromolar FPP and 50 micromolar C14 IPP. Initiate the reaction in buffer A with 0.5 millimolar magnesium chloride at 22 to 30 degrees Celsius. Withdraw 15 microliter samples from the reaction at zero, two, four, and six hours following immediate quenching of the reaction by the addition of 15 microliters of buffer A supplemented with 20 millimolar EDTA.
Then, add one milliliter of water-saturated 1-butanol and vortex thoroughly to extract the reaction products. The protocol can be stopped here, and the samples can be kept for later reading. Next, add the scintillation cocktail to the samples.
Using a scintillation counter, quantitate reaction products in the butanol phase encompassing C14 together with radioactivity of 15 microliters of the reaction, representing the total radioactivity. Finally, calculate the net C14 IPP incorporation at each time point by calculating the percent utilized from the total C14 IPP concentrations and plot the results as a function of time. An increase in the net C14 IPP incorporation is expected with time.
The samples obtained at each purification step are shown here. This SDS-PAGE analysis shows the stepwise purification of DHDDS, resulting in a highly-purified product. This chromatogram represents the results of analytical size exclusion chromatography of the purified enzyme, revealing that the protein is only observed as a homodimer.
Here, the arrow indicates the void volume. A representative time-dependent activity assay reveals that C14 IPP incorporation clearly rises over six hours, verifying that the purified enzyme is functional. Once mastered, this simple preparation can be done in two to three days, resulting in a highly pure and active enzyme.
After watching this video, you should have a good understanding of how to overexpress and purify human cis-prenyltransferase from E.coli and to measure its activity in a time-dependent manner.
