Name: overexpression and purification of human cis-prenyltransferase in escherichia coli
Uploaded: 2017-08-03T15:00:00
Description: Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

This work was funded by the Israel Science Foundation's Center for Research Excellence (I-CORE) in Structural Cell Biology (1775/12) and the Israel Science Foundation grants 1721/16 and 2338/16 (Y.H.), and 825/14 (D.K.). The support of the Fields Estate Foundation to D.K. is highly appreciated. This work was performed by Ilan Edri and Michal Goldenberg in partial fulfillment of the M.D. thesis requirements of the Sackler Faculty of Medicine, Tel Aviv University.

1 . Cloning of cis-PT for Overexpression in E. coli
<ol>
	<li>Obtain pET-32b expression vector, designed for cloning and high-level expression of protein sequences fused with the 109aa thioredoxin (TRX) protein 17, and E. coli codon-optimized 18 coding sequence of full-length cis-PT.</li>
	<li>Take care to have a TEV-protease (tobacco etch virus protease) cleavage site (ENLYFQ/G, where &#34;/&#34; indicates the cleavage point) <sup clas...

<ol>
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The protocol described here for purification of functional human DHDDS in E. coli cells is simple and efficient, allowing one to overexpress and purify the protein in 3 - 4 days once a suitable construct is available. Such protocols for protein purification are of special significance given the breakthroughs in genome sequencing, which provided plethora of information regarding the genetics of many diseases 18, thereby requiring the development of high-throughput methods to characterize p...

Prenyltransferases are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions 1,2. Z-type enzymes catalyze the formation of cis double bonds from the condensation reaction, whereas E-type enzymes catalyze trans double bond formation 3. cis-Prenyltransferases (cis-PT, Z-type enzymes) are classically classified according to their product chain length into short-chain (C15), medium-chain (C50-55), and long-chain (C70-120) 4. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP) 1,5,6. This results in the formation of dehydrodolichyl diphosphate, a C55-100polyprenyl diphosphate serving as a precursor for dolichylpyrophosphate, the glycosyl carrier molecule involved in N-linked protein glycosylation 1. Among Ashkenazi Jews, a missense non-conservative mutation (K42E) in DHDDS results in autosomal recessive retinitis pigmentosa 7,8. Therefore, the present protocol was developed in order to acquire purified DHDDS suitable for mechanistic studies.
Escherichia coli is considered the most convenient and cost-effective host for recombinant protein expression, and is therefore also the most frequently used host. However, when one attempts to heterologously overexpress proteins in E. coli, protein-specific considerations should be made. Obtaining properly folded, active recombinant proteins from E. coli, is not a simple matter due to the distinct properties of different proteins. Numerous approaches have been developed to overcome these hurdles. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. Of note, a previous attempt to overexpress yeast cis-PT without protein fusion was unsuccessful due to complete insolubility even in the presence of detergent 12. The described protocol is simple, cost-effective, time sparing and allows one to obtain DHDDS preparations suitable for mechanistic studies. Given the homology of cis-PT among different species, we suggest that this protocol may be applied for other eukaryotic cis-PT as well.

<table width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

General overview of the construct used here and the purification process are shown in Figure 1. The samples obtained at each purification step are shown in Figure 2. This SDS-PAGE analysis shows the stepwise purification of DHDDS, resulting in a highly purified product. Figure 3 shows the results of analytical SEC of the purified enzyme, revealing that the protein is only observed as a homodimer. <st...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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.

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