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.
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.
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.
1 . Cloning of cis-PT for Overexpression in E. coli
2 . Overexpression of Human DHDDS in E. coli
3 . Purification of Human DHDDS
4 . Analytical Size-exclusion Chromatography (SEC)
5 . Enzyme Kinetics – Time-dependent Activity 15,16,17
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. Figure 4 shows a representative time-dependent activity assay. 14C-IPP incorporation clearly rises over 6 h, verifying that the purified enzyme is functional.
Figure 1: Human DHDDS Cloning and Purification. (A) The construct used in this study. (B) Outline of the human DHDDS purification protocol described here. Please click here to view a larger version of this figure.
Figure 2: Purification of Human DHDDS. SDS-PAGE analysis of human DHDDS affinity purification steps. Lane 1, molecular-weight marker (kDa); lane 2, crude extract; lane 3, Co2+-IMAC flow-through. Arrow indicates the TRX-DHDDS fusion protein; lane 4, Co2+-IMAC eluate; lane 5, protein-TEV protease mixture following overnight incubation; lane 6, Co2+-IMAC flow-through following TEV cleavage; lane 7, Co2+-IMAC eluate following TEV cleavage; lane 8, purified human DHDDS (indicated by an arrow) following size-exclusion chromatography. Please click here to view a larger version of this figure.
Figure 3: Analytical SEC of Purified Human DHDDS. Tryptophan fluorescence was monitored as described in the protocol. According to the calibration curve of this column, DHDDS forms a homodimer (77.4 kDa). The arrow indicates the void volume. Please click here to view a larger version of this figure.
Figure 4: Time-dependent Activity of Purified Human DHDDS. In vitro activity of purified human DHDDS in the reaction with 10 μM FPP and 50 μM 14C-IPP as substrates is expressed as IPP incorporation per protein (mol/mol). Please click here to view a larger version of this figure.
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 pathogenic mechanisms at the protein level 19.
To overcome common pitfalls in heterologous protein overexpression and purification, several measures were taken here, which are critical to successfully obtain soluble and functional proteins. First, DHDDS was overexpressed as a TRX-fusion. TRX facilitates the fusion protein folding and increases the fusion protein solubility, preventing inclusion body formation 20. Next, to increase protein yield, the DNA construct was codon optimized for expression in E. coli 21. Finally, to further reduce the likelihood of inclusion body formation, expression of the protein was induced at 16 °C to slow down the protein synthesis rate, likely assisting protein folding 22.
Taking into consideration the homology of cis-PT among different species, we suggest that this protocol may be applied for other eukaryotic cis-PT as well 1. Using the current protocol as a starting point, and given the general guidelines critical for DHDDS expression, this method can be modified for optimal expression of different cis-PTs. For example, one may attempt different fusion partners (such as maltose-binding protein) or further optimize the culture conditions for the specific protein to be studied.
With their biomedical and biotechnological significance 1,7,8, future usage of the described protocol to obtain large quantities of purified functional DHDDS, together with other cis-PT, allowing the pursuit of their structural and functional characterization. For example, further molecular studies of DHDDS will resolve the mechanisms underlying DHDDS-related retinitis pigmentosa, potentially leading to discovery and development of novel therapeutic approaches. In addition, as prokaryotic cis-PTs are involved in bacterial wall synthesis, this group of enzymes potentially forms novel targets for new antibacterial agents. Indeed, thorough characterization of the eukaryotic and prokaryotic enzymes studies may allow the rational development of antimicrobial drugs selective for prokaryotic cis-PT. Finally, as natural rubber is synthesized by members of the cis-PT family, the approach described here may find future use in natural rubber industrial synthesis.
The authors have nothing to disclose.
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.
pET-32b | Novagen | 69016-3 | |
T7 Express lysY Competent E. coli (High Efficiency) | NEB | C3010I | |
cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | 11873580001 | |
TALON-superflow resin | GE Healthcare | 28-9574-99 | |
HiPrep 26/10 desalting column | GE Healthcare | 17508701 | |
HiLoad 16/60 superdex-200 | GE Healthcare | 28989335 | |
superdex-200 increase 5/150 GL | GE Healthcare | 28990945 | |
14C-Isopentenyl pyrophosphate | Perkin-Elmer | NEC773050UC | |
trans,trans-Farnesyl pyrophosphate | Sigma | 44270-10MG |