Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies

Diacylglycerol kinases (DGKs) are lipid kinases that modulate the levels of lipid second messengers, diacylglycerol and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. DGKα consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal catalytic domain (DGKα-CD). To date, however, no structures of mammalian DGKs including their CDs have yet been reported, impeding our understanding on the catalytic mechanism of DGKs and the rational structure-based drug design. Here we attempted to produce DGKα-CD or a full-length DGKα using bacterial and baculovirus-insect cell expression system for structural studies. While several DGKα-CD constructs produced using both bacterial and insect cells formed insoluble or soluble aggregates, the full-length DGKα expressed in insect cells remained soluble and was purified to near homogeneity as a monomer with yields (1.3 mg/mL per one L cell culture) feasible for protein crystallization. Following enzymatic characterization showed that the purified DGKα is in fully functional state. We further demonstrated that the purified enzyme could be concentrated without any significant aggregation, and characterized its secondary structure by circular dichroism. Taken together, these results suggest that the presence of N-terminal regulatory domains suppress protein aggregation likely via their intramolecular interactions with DGKα-CD, and demonstrate that the baculovirus-insect cell expression of the full-length form of DGKα, not DGKα-CD alone, represents a promising approach to produce protein sample for structural studies of DGKα. Thus, our study will encourage future efforts to determine the crystal structure of DGK, which has not been determined since it was first identified in 1959.

Diacylglycerol kinase a is the first-cloned DGK isozyme in mammals (Sakane et al., 1990) and has amino-terminal regulatory domains including EF-hand motifs and C1 domains, and a carboxyl-terminal CD (Fig. 1A). Recently, increasing attention has been paid to DGKa as a potential target for anti-cancer treatments including cancer immunotherapy (Dominguez et al., 2013;Purow, 2015;Sakane, Mizuno & Komenoi, 2016;Liu et al., 2016;Noessner, 2017). Expression of DGKa has been reported to be upregulated in melanoma cells (but not in noncancerous melanocytes) (Yanagisawa et al., 2007), lymphoma (Bacchiocchi et al., 2005), hepatocellular carcinoma (Takeishi et al., 2012), breast cancer cells (Torres-Ayuso et al., 2014), and glioblastoma cells (Dominguez et al., 2013) where DGKa promotes cancer cell survival, proliferation, migration, and invasion (Merida et al., 2017). siRNA knockdown of DGKA or inhibition of DGKa by small molecule inhibitors for DGKs, R59022 and R59949, has detrimental effects on the proliferation of glioblastoma cells, melanoma, breast cancer, and cervical cancer cells (Yanagisawa et al., 2007;Dominguez et al., 2013). In T-lymphocytes, on the other hand, DGKa is appreciated as a critical attenuator for immune response. DGKa is highly expressed in T-cells and decreases membrane DG levels required for RasGRP1-dependent activation of the Ras-Erk pathway (Jones et al., 2002). Furthermore, in vitro and in vivo studies have uncovered that DGKa is responsible for T-cell hyporesponsive state known as anergy state (Olenchock et al., 2006;Zha et al., 2006).
Using a high-throughput DGK assay, we have recently identified a novel DGKa-selective inhibitor, CU-3, and revealed that this compound targets the CD of DGKa (Liu et al., 2016). Indeed, this compound not only induced the apoptosis of HepG2 hepatocellular carcinoma and HeLa cervical cancer cells as observed for other DGK inhibitors with lower-selectivity (Dominguez et al., 2013), but also enhanced the production of interleukin-2 in Jurkat T cells (Liu et al., 2016), illustrating a double-strike effect of DGKa inhibitors potentially utilized for cancer immunotherapy (Noessner, 2017). However, despite these biological and biomedical importance, no structure has been determined for the CDs of any mammalian DGKs, thus impeding the detailed understanding of DGK catalytic machinery and substrate binding sites as well as the development and optimization of effective DGKa inhibitors.
One of the greatest challenges for the structure determination of DGK isozymes lies in producing enough and soluble proteins suitable for protein crystallization, as illustrated in previous studies (Takahashi et al., 2012;Petro & Raben, 2013). Although intensive efforts have been made by Petro & Raben (2013) to express and purify the full-length and CD of porcine DGKa using bacterial expression system with expression-tags (glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX)) for solubility enhancement, all the expressed DGKa constructs formed inclusion bodies or soluble aggregates, likely due to the inability of bacterial translational and folding machineries. Cell lysates were separated into supernatant and insoluble pellets and subjected to SDS-PAGE (10%) followed by immunoblot analysis using anti-DGKa antibody. (C) SDS-PAGE (10%) analysis of fractions from Ni 2+ -affinity purification. Separated proteins were stained with Coomassie blue staining. (D) Elution profile of DGKa from size exclusion chromatography. Fraction numbers used for following SDS-PAGE analysis are labeled. The inset shows the calibration of gel-filtration column using protein standards of known molecular weight (thyroglobulin (670 kDa), -globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa)). Partition coefficient (K av ) was calculated from the formula, where V E is the retention volume of each sample, V T is the total column volume (120 mL), and V 0 is the void volume of the column (44 mL), respectively. K av was plotted against the molecular weight of proteins and linear regression analysis was conducted. (E) SDS-PAGE (10%) analysis of DGKa purified using size-exclusion chromatography.
Full-size  DOI: 10.7717/peerj.5449/ fig-1 To overcome these problems, here we have taken advantage of the baculovirus-insect cell expression system to express a full-length DGKa in soluble form. DGKa expressed in the insect cells was then purified by a series of column chromatography, and the purified protein was found to be a monomer in solution. Successful purification of DGKa also allowed us to characterize enzymatic, inhibitory and structural properties of DGKa in vitro. Taken together, these results provide promising evidence that the baculovirus-insect cell expression system is better suited to produce DGKa for in vitro functional and structural studies.

Bacterial expression and purification of DGKa-CD
Multiple-constructs approach with different N-and C-terminal boundaries (Gräslund et al., 2008), and several N-terminal fusion-tags (GST, MBP, and small ubiquitin-like modifier (SUMO)) was applied for bacterial expression of DGKa-CD.
To prepare GST-fused constructs, the DNA sequences of DGKa-CD (S332-G722, D344-G722, D369-G722, S332-S735, D344-S735, D369-S735), flanked by BamHI and SalI restriction sites were amplified by PCR from the full-length cDNA for human DGKa, inserted into a pGEX-4T-2 vector (GE Healthcare Life Science, Little Chalfont, UK) and the resulting plasmids were used to transform Escherichia coli strain Rosetta2 (DE3) (Novagen, Madison, WI, USA). The protein construct contained a thrombin-cleavable GST-tag before the DGKa-CD sequence. Cells were cultured in LB media at 37 C until OD 600 reached 0.6-0.8. Expression of the recombinant protein was then induced by adding 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG), and the bacterial culture was continued at 16 C for overnight. Bacteria harvested by centrifugation were suspended in a lysis buffer (50 mM sodium phosphate, pH 8.0, containing 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) and lysed by sonication on ice. Protease inhibitors (20 mg/mL aprotinin, 20 mg/mL leupeptin, 20 mg/mL pepstatin, 1 mM soybean trypsin inhibitor) were added immediately before sonication. To evaluate expression and solubility of the expressed proteins, soluble and insoluble fractions were separated by centrifugation at 15,000Âg for 10 min and subjected to SDS-PAGE (10%) followed by Coomassie Brilliant Blue (CBB) staining and immunoblot analysis using anti-GST monoclonal antibody (B-14; Santa Cruz Biotechnology, Dallas, TX, USA). The immunoreactive bands were visualized using peroxidase-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and the ECL Western blotting detection system (GE Healthcare Life Science, Little Chalfont, UK).

Expression of DGKa in insect cells using baculovirus expression vector system
The construct of DGKa-CD (D364-S735) or full-length DGKa with N-terminal His Â6 tag was PCR-amplified and cloned into the pOET3 vector (Oxford Expression Technologies, Oxford, UK) via SalI/NotI sites. The resulting DNA sequences were verified to be correct by DNA sequencing. The flashBAC system (Oxford Expression Technologies, Oxford, UK) was used to generate a recombinant baculovirus and the virus stock was amplified by several rounds of infection of Sf9 cells cultured in Sf-900 II serum free medium (Invitrogen, Carlsbad, CA, USA) at a low multiplicity of infection (MOI). Plaque assays were performed to determine titers of the amplified virus stocks. Both DGKa-CD and full-length DGKa were expressed in Sf9 cells by infecting the cells (at 2 Â 10 6 cells/mL) with the baculovirus stock at MOI of 2. Cells were cultured at 28 C with shaking for 24 h and pelleted by centrifugation at 1,500Âg, 4 C for 20 min and washed with sterile phosphate buffered saline before storage at -80 C.

In vitro DGKa activity assay
Activity of full-length DGKa was determined using the octyl-b-D-glucoside mixed micelle assay combined with the ADP-Glo TM kinase assay kit (Promega, Madison, WI, USA), as previously described (Sato et al., 2013;Liu et al., 2016). Briefly, the substrate micelle mixture containing 50 mM n-octyl-b-D-glucoside (Dojindo, Kumamoto, Japan), 10 mM (27 mol%) phosphatidylserine (PS; Sigma-Aldrich, St. Louis, MO, USA), 2 mM (5.4 mol%) 1,2-dioleolyl-sn-glycerol (DG; Sigma-Aldrich, St. Louis, MO, USA), 0.2 mM adenosine 5'-triphosphate (ATP) in a final buffer consisting of 50 mM MOPS, pH 7.4, 100 mM NaCl, 20 mM NaF, 10 mM MgCl 2 , 1 mM CaCl 2 was mixed with 5 mL of purified DGKa to initiate enzymatic reaction. The reaction mixtures were incubated at 30 C for 30 min. Subsequently, 25 mL of ADP-Glo reagent was added and incubated at room temperature for 40 min to terminate the enzyme reaction and deplete the remaining ATP. Kinase Detection Reagent (50 mL) was then added to convert the ADP produced from the kinase reaction into ATP for a luciferase/luciferin reaction. The reaction was performed at room temperature for 40 min and the luminescence from the luciferase/luciferin reaction was measured with a GloMax microplate reader (GloMax; Promega). A standard curve for ADP was generated by fitting a various concentration of ADP ranging from 25 to 200 mM and the corresponding luminescence signals relative luminescence unit by linear regression, and was used to convert the luminescence intensities from DGKa reaction into ADP concentrations. To determine kinetic constants, the activity assay was performed under a series of concentrations of ATP (20 mM-1 mM) and DG (0-5.4 mol%), respectively. DGKa purified by size exclusion chromatography was added to 100 ng for each reaction and the assays were done in triplicate for each ATP or DG concentrations. The K m value was obtained by fitting the kinase activity of DGKa with the Michaelis-Menten equation using Prism 7 (GraphPad Software, La Jolla, CA, USA). To test the calcium dependency of DGKa activity, the enzyme activity was measured under the conditions containing either EGTA (3 mM) or CaCl 2 (0.6 mM).

Inhibitor activity assay
Inhibitory activity of a previously identified inhibitor, CU-3 (Liu et al., 2016), against DGKa was measured with the octyl-b-D-glucoside mixed micelle assay followed by the ADP-Glo assay. A concentration series of CU-3 (0.02-10 mM) was incubated with the purified DGKa for 30 min at room temperature before adding to a reaction mixture for the assay. Half maximal inhibitory concentration (IC50) was determined by fitting the CU-3 dependent decrease of DGKa activity with the variable slope model in Graphpad Prism 7 software.

Circular dichroism spectroscopy
Circular dichroism spectrum were recorded at ambient conditions between 190 and 250 nm on a Jasco J-805 spectrometer (JASCO Corporation, Tokyo, Japan) using a cell with path length of 0.2 mm, 20 nm/min scan speed and a bandwidth of 1 nm. DGKa was prepared at 0.32 mg/mL (3.75 mM) in 20 mM Tris-HCl buffer, pH 7.5, 10 spectra were averaged and a spectrum obtained for the buffer was subtracted. Spectral data were analyzed using the program Contin-LL (Provencher & Glöckner, 1981) suited in the DICHROWEB platform (Whitmore & Wallace, 2004).

A full-length form of DGKa was expressed in baculovirus-infected insect cells and purified as a monomer
We have previously reported that DGKa-CD possess enzymatic activity comparable to that of the full-length enzyme when expressed in COS-7 cells (Sakane et al., 1996), indicating that its substrate (ATP and DG) binding sites locate in the CD and DGKa-CD is an essential target for inhibitor development. Full-length DGKa also contains cysteinerich C1 domains (Fig. 1A), which might be detrimental for correct folding in heterologous expression hosts. Therefore, we have first attempted to express DGKa-CD in E. coli by revamping the previous approach by Petro & Raben (2013). In addition to N-terminal GST and MBP-tags, which were previously utilized (Petro & Raben, 2013), we have used Sumo domain fusion-tag for the enhancement of expression and solubility (Butt et al., 2005;Marblestone et al., 2006). To further increase the chance for expression of soluble proteins, we have also applied a multiple-construct approach (Gräslund et al., 2008) to prepare DGKa-CD constructs which have different N-and C-terminal boundaries (S332-G722, D344-G722, D369-G722, S332-S735, D344-S735, D369-S735). Each of those constructs was fused with the GST, MBP, and Sumo-tags. Despite our efforts, those constructs resulted in either insoluble inclusion body formation (with GST-tag), or insufficient translation and proteolytic degradation (with MBP-tag), or soluble microscopic aggregation (with Sumo-tag) (Fig. S1).
To circumvent the difficulty associated with bacterial expression system, we have used baculovirus-infected Sf9 cells to produce DGKa-CD. The construct of DGKa-CD (D364-S735) with N-terminal His Â6 tag was cloned into pOET3 transfer vector harboring the late AcMNPV p6.9 promoter, which provides earlier expression compared to the polyhedrin promotor. The recombinant DGKa-CD was expressed in cultured insect cells using the flashBAC baculovirus vector expression system, and subsequently purified from cell-lysates using Ni-affinity chromatography (Fig. S2A). Following size-exclusion chromatography on a Superdex 200, however, demonstrated that DGKa-CD formed soluble aggregates eluting in the void volume of the column (Fig. S2B).
In our early studies, a native form of full-length DGKa has been purified from porcine thymus and this full-length form was found to be catalytically competent Sakane et al., 1991). We therefore set to produce full-length DGKa (aa 1-735) using the same baculovirus expression system used for DGKa-CD. As expected, the vast majority of DGKa remained in soluble form after cell lysis, as shown by immunoblot analysis (Fig. 1B). Ni-affinity chromatography was conducted to purify DGKa from the cell lysis supernatant, and relatively pure DGKa was eluted in fractions containing 50 and 100 mM imidazole (Fig. 1C). To further purify DGKa, we next performed size-exclusion chromatography on a Superdex 200 column. Because DGKa bears calcium-binding EF-hand motifs and a magnesium ion was predicted to bind to the CD (Abe et al., 2003), we added 3 mM CaCl 2 and 3 mM MgCl 2 in the equilibration buffer. DGKa eluted as a single peak at 73.5 mL retention volume (Fig. 1D), which corresponds to the molecular mass of 77 kDa, based on a calibration curve obtained with molecular mass standard proteins. This result indicates that DGKa exists as a monomer in solution. DGKa was purified to near homogeneity (Fig. 1E) and the yield was approximately 1.3 mg per one L of Sf9 cell culture.

Kinase activity assay and inhibitory assay for the purified DGKa
To test whether the purified DGKa is catalytically active, we conducted the octyl-b-Dglucoside mixed micelle assay combined with a luminescence-based assay that measures ADP produced in a kinase reaction (Sato et al., 2013;Liu et al., 2016). DGKa purified from the size-exclusion chromatography was found to exhibit kinase activity with peak fractions having the maximum activity ( Fig. 2A). We have previously demonstrated that DGKa activity, which has been purified from porcine thymus or expressed in COS-7 cells, is enhanced by Ca 2+ binding to its two N-terminal EF-hand motifs (Sakane et al., 1990(Sakane et al., , 1991Yamada et al., 1997). As predicted, the purified DGKa exhibited significantly reduced activity when the bound calcium ions were chelated with 3 mM EGTA (Fig. 2B). Furthermore, no significant changes of the activity were observed after storage of the purified DGKa at 4 C for at least 3 months.
We also determined the kinetic parameters of DGKa for ATP and DG to assess the catalytic properties of the purified DGKa. ATP-dependent increase of the kinase activity Five microliters from each fraction containing 38.5-363 ng of DGKa was added for a reaction and the following details are described in "Materials and Methods." Luminescence values are presented as relative luminescence unit (RLU) over background signals from a well containing a buffer (20 mM Tris-HCl, pH 7.4, 0.2M NaCl, 3 mM CaCl 2 , 3 mM MgCl 2 , 0.5 mM DTT, and 5% glycerol) used for size-exclusion chromatography. (B) Calcium-dependent activity of the purified DGKa. The luminescence-based DGK activity assay was conducted using 150 ng of DGKa in the presence of CaCl 2 (0.6 mM) and EGTA (3.6 mM). Purified DGKa was pre-incubated with 3 mM EGTA for 30 min on ice to chelate CaCl 2 contained in a buffer used for size exclusion chromatography, and concentrated EGTA was also added into the reaction mixture at a final concentration of 3.6 mM. Measured luminescence values of DGKa in the presence of CaCl 2 or EGTA were subtracted with each negative control (CaCl 2 or EGTA) and data shown are mean ± SD for triplicate measurements. was observed (Fig. 3A) and the K m value was determined to be 0.24 ± 0.03 mM (Table 1), comparable with those obtained with DGKa from porcine thymus (0.1 mM) (Sakane et al., 1991) or DGKa expressed in COS-7 cells (0.1-0.25 mM) (Sato et al., 2013;Liu et al., 2016). The activity was also increased in a DG-concentration dependent manner (Fig. 3B) and the K m value of 1.1 ± 0.21 mol% (Table 1) was consistent with those from our previous studies (3.3 mol% with DGKa purified from porcine thymus (Sakane et al., 1991), 1.9-3.4 mol% with DGKa expressed in COS-7 cells (Sato et al., 2013;Liu et al., 2016)). For both cases, compared to our previous study using crude lysates of mammalian cells (Sato et al., 2013;Liu et al., 2016), the relative activity increased nearly 50-fold when the purified DGKa was used. Furthermore, the kinase activities of our purified DGKa (1 to 2 nmol/min/mg) is comparable to those obtained with DGKa from porcine thymus (2.4 nmol/min/mg) (Sakane et al., 1991). These results demonstrate that the purified DGKa is in a fully functional state and stable during purification and storage. We next measured the inhibitory activity of CU-3, a previously identified DGKa inhibitor (Liu et al., 2016). CU-3 is an ATP competitive inhibitor with an IC 50 value

Structural characterization of the purified DGKa
We also found that DGKa solution could be concentrated using a centrifugal filter without any significant loss of the protein. Concentrated DGKa remained as a monomer as demonstrated by a size-exclusion chromatography (Fig. S3). Using the concentrated DGKa (0.32 mg/mL), we characterized the secondary structure using circular dichroism spectroscopy. The circular dichroism spectrum of DGKa and following analysis indicates that DGKa is well-folded and contains certain amounts of a-helical (18.9%) and b-strand (27.4%) structures (Fig. 5), further demonstrating that the expression of a full-length DGKa, not a solo CD, in the baculovirus-infected insect cells is suitable for producing a natively folded and active form of DGKa.

DISCUSSION
Diacylglycerol kinases are a family of multi-domain lipid kinase that regulate a variety of cellular process Merida, Ávila-Flores & Merino, 2008;Shulga, Topham & Epand, 2011), and DGKa has recently emerged as a novel therapeutic Figure 5 Secondary structure of the purified DGKa. Circular dichroism spectrum of DGKa measured at ambient conditions between 190 and 250 nm on a Jasco J-805 spectrometer. DGKa was prepared at 0.3 mg/mL in 20 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 3 mM MgCl 2 , 3 mM CaCl 2 , 5 % glycerol. The analysis of the circular dichroism spectrum using the program Contin-LL (Provencher & Glöckner, 1981) suited in the DICHROWEB platform (Whitmore & Wallace, 2004) showed the presence of both a-helical (18.9 %) and β-strand ( target for cancer immunotherapy (Dominguez et al., 2013;Purow, 2015;Sakane, Mizuno & Komenoi, 2016;Liu et al., 2016;Noessner, 2017). However, no structural information of DGKs, especially their CD, is available. This is largely because the procedure for large scale production of recombinant DGKs in their soluble and homogeneous form, a prerequisite for protein crystallization, is not well-established.
Here we have used the baculovirus-insect cell expression system to produce a full-length form (DGKa), and investigated the enzymatic and structural properties in vitro. Petro & Raben (2013) have made significant efforts to express and purify a pig DGKa and DGKa-CD using bacterial expression system with several fusion tags (GST, TRX, and MBP), a set of bacterial chaperons, or in vitro refolding. Despite their pursuit, expressed DGKa constructs either formed inclusion bodies or soluble microscopic aggregates. We have also used E. coli cells to produce DGKa-CD with several N-terminal fusion tags (GST, Sumo, and MBP) and with different N-and C-terminal boundaries. While both MBP-and Sumo-fused DGKa-CD remained in a soluble fraction after cell-lysis and Ni-affinity chromatography (Figs. S1A and S1B), those DGKa-CD with fusion-tags eluted in the void volume of Superdex 200 column (Fig. S1C). When expressed with MBP, an elution fraction from the Ni-affinity chromatography also contained additional smaller bands along with MBP-fused DGKa-CD (Fig. S1A), which could be due to insufficient translational ability of E. coli for producing eukaryotic proteins, as previously suggested (Petro & Raben, 2013). Because baculovirus-insect cell expression system has both the capacity to produce recombinant proteins at a large scale and the capability to provide eukaryotic protein expression machineries, we next utilized this system to produce DGKa-CD. The protein expressed in Sf9 cells was soluble, however, contrary to our expectation, the protein formed soluble aggregates, which eluted in the void volume (Fig. S2). These results indicate that the only CD has a tendency to self-aggregate upon isolation, possibly due to its intrinsic characteristics that recognize DG embedded in plasma membrane, and suggest that the only CD is not suitable for structural studies even if it is expressed using a eukaryotic expression system.
In contrast to the CD, full-length DGKa elutes in a relatively sharp peak of sizeexclusion chromatography and remains as a monomer when it is assumed to have a globular shape ( Fig. 1C; Fig. S3). Such production of a full-length DGKa in a soluble and monomeric form using the baculovirus insect cell expression system holds promise for the preparation of DGKa sample suitable for protein crystallization screening. DGKa consists of the N-terminal regulatory domains including EF-hand motifs and C1 domains, and the C-terminal CD. This suggests that DGKa adopts a compact globular structure rather than an elongated one. YegS (a putative bacterial lipid kinase) (Bakali, Nordlund & Hallberg, 2006), a bacterial DgkB (Miller et al., 2008), and a human sphingosine kinase (SphK1) (Wang et al., 2013) have been successfully purified and their crystal structures have been reported (Bakali et al., 2007;Miller et al., 2008;Wang et al., 2013). Although all of those lipid kinases are homologous to mammalian DGKs and belong to a protein family PF00781 (DAGK_cat), they do not possess N-terminal regulatory domains. This might explain why the N-terminal domain of DGKa is required to obtain the protein as a soluble monomer. Interestingly, previous studies by us and others have suggested the presence of intramolecular interactions between the N-terminal regulatory domains and the CD (Merino et al., 2007;Takahashi et al., 2012). It is reasonable to surmise that a potential aggregation-prone surface of the CD of DGKa is intra-molecularly masked by the N-terminal regulatory domains including recoverin homology, EF-hand motif, and C1 domains.
Enzymatic characterization of DGKa reveals that K m values to ATP (0.24 mM) and DG (1.1 mol%) are very similar to those obtained using DGKa partially purified from porcine thymus (0.1 mM for ATP and 3.3 mol% for DG, respectively) (Sakane et al., 1991) or DGKa expressed in COS-7 cells (0.1-0.25 mM for ATP and 1.9-3.4 mol% for DG, respectively) (Sato et al., 2013;Liu et al., 2016), further demonstrating the effectiveness of baculovirus insect cell expression system for producing DGKa not only in soluble and homogeneous form, but also in its active one.
In summary, this study demonstrates that the production of full-length DGKa, not DGKa-CD alone, using the baculovirus-insect cell expression is a very promising approach to produce DGKa samples for future in vitro structural and functional studies. Firstly, DGKa has been purified by Ni-affinity and size-exclusion chromatographies to near-homogeneity, and purified DGKa remains in soluble and monomeric form, and can be concentrated without any significant loss of the protein, which are prerequisites for protein crystallization. Purified DGKa sample, however, still contains slight amounts of contaminant proteins which might non-specifically bind to DGKa. Further modification and optimization of the protein construct and purification conditions must be required. Secondly, the obtained yield of DGKa, 1.3 mg per one L cell culture, is enough to initiate crystal screening. Thirdly, the purified DGKa is catalytically competent. The measured kinase activity and the K m values to ATP and DG are comparable to those obtained with native form of DGKa partially purified from porcine thymus and DGKa expressed in mammalian cells.

CONCLUSION
We demonstrate that the baculovirus-insect cell expression of the full-length form of DGKa, not DGKa-CD alone, represents a promising approach to produce protein sample suitable for structural studies of DGKa. We believe that this study will encourage future pursuits to determine crystal structures of mammalian DGKs that has still remained enigmatic for almost 60 years since its identification (Hokin & Hokin, 1959).