Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins.


Introduction
The baculovirus expression system (BES) is widely used for the production of vertebrate proteins or vaccines in insect cells or larvae. The BES is less expensive than mammalian cell expression systems, has a convenient protein production method, and has a short production time [1]. The most useful feature of the BES is that it leads to the expression of correctly folded and post-translationally modified proteins at a similar level to mammalian expression systems [2,3]. The BES uses a polyhedrin promoter with strong activity. However, the production efficiency of a foreign protein using this polyhedrin promoter is not as high as that of native polyhedrin [4]. Therefore, various attempts have been made to increase the production efficiency of foreign proteins, such as the addition of transcriptional enhancers or fusion with a carrier protein as a "fusion partner". The fusion partner is frequently highly expressed in host cells, which not only enhances the expression level of the fusion protein, but also favors the purification of the fusion protein [5]. However, these methods have not been applied to all proteins; thus, it is necessary to remove the fusion partner to prevent structural and functional changes to the target protein [6,7]. Therefore, enzymes such as a TEVp protease [8], TAGZyme [9], or enterokinase [10] should be used, although they have the disadvantage of requiring an additional process for the removal

Construction of Transfer Vector
The Ssp DnaB mini-intein gene was amplified from the pTWIN1 plasmid (New England Bio labs, Ipswich, MA, USA) with the primers His6-SspDnaB-F and SspDnaB-R ( Table 1). The PCR products were directly cloned into the pMD20-T-vector (TaKaRa, Kusatsu, Shiga, Japan) and digested with EcoR I and Pst I and were then subsequently cloned into the corresponding restriction sites of the pBacPak9 vector (Clontech, Mountain View, CA, USA) to construct pB9-His6-SspDnaB. The enhanced green fluorescent protein (EGFP) gene was amplified from a pEGFP-N1 plasmid (Clontech, Mountain View, CA, USA) with the primers EGFP-F and EGFP-R ( Table 1). The PPV VP2 gene was amplified from PPV with the primers PPV-VP2-F and PPV-VP2-R ( Table 1). The CSFV E2 gene (CSFV-E2-∆TMR) without transmembrane region (TMR) was amplified from CSFV with the primers CSFV-E2-F and CSFV-E2-R ( Table 1). The PCR products were directly cloned into the T-vector and digested with Nco I and Pst I and were subsequently cloned into the corresponding restriction sites of the pB9-His6-SspDnaB vector to construct pB9-His6-SspDnaB-EGFP, pB9-His6-SspDnaB-PPV-VP2 and pB9-His6-SspDnaB-CSFV-E2 ( Figure 1A). To generate control recombinant baculoviruses, the cloned PCR products in the T-vector were digested with BamH I and Pst I and subsequently cloned into the corresponding restriction sites of the pBacPak9 vector to generate pB9-EGFP, pB9-PPV-VP2 and pB9-CSFV-E2 ( Figure 1).

Name of Primer
Primer Sequence * The restriction site incorporated into each oligomer is underlined. The forward PCR primer designed for cloning a given target protein into the system must include the Nco I site and the subsequent CA nucleotide sequence before the annealing portion on the target, which allows for the construction of a proper in-frame fusion.  PCR amplification of each of the DNA fragments that were cloned into the T-vector; the PCR products were sequentially cloned into the transfer vector pBacPAK9. Finally, each constructed transfer vector was co-transfected into Bm5 cells with bBmGOZA to generate each recombinant BmNPV.

Measurement of Fluorescence
To measure the fluorescence intensity, rBm-His6-SspDnaB-EGFP was infected into Bm5 cells. The infection was carried out with 1 × 10 6 cells per well in 6-well plates that were infected with a multiplicity of infection (MOI) of 5 PFU/cell. Infected cells were collected at 24 h intervals from 24 to 120 hours post-infection (h.p.i) and washed with ice cold PBS. The lysate was prepared by incubating the cells with 1 mL of a lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 7.0, protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA)) for 30 min on ice followed by sonication, and then, 2 mL of PBS was added. Fluorescence measurements were performed at room temperature in quartz cuvettes with a minimum test volume of 3 mL. The fluorescence intensity of the resulting mixture samples was measured using a K2TM fluorescence spectrometer (ISS, Inc., Champaign, IL, USA) with an excitation filter of 450 nm and emission filter of 510 nm. A minimum of three trials were conducted as previously described [21].

Immuno-Fluorescence Assay
Bm5 cells were cultured on sterile cover slips (placed in 6-well plates) and infected with rBm-His6-SspDnaB-EGFP, rBm-His6-SspDnaB-PPV-VP2, and rBm-His6-SspDnaB-CSFV-E2 at 5 MOI. After 3 days of infection, cells were fixed with cold methanol for 3 min at −20 • C, rinsed with PBS, and blocked with 2% bovine serum albumin for 30 min at 37 • C. Then, the cells were washed three times with PBS. Subsequently, the cells were incubated with specific antibodies against E2 or VP2 for 1 h. After washing the cells with PBS, they were incubated with an Alexa Fluor ® 488-conjugated goat antibody (Abcam, Cambridge, UK). Nuclei were stained with PBS containing 10 µg/mL DAPI (Sigma-Aldrich, Saint Louis, MO, USA), and the cells were washed thoroughly. Cover slips were mounted on glass slides with one drop of 50% glycerol in PBS and air dried for 15 min. Visualization and localization of the nucleus and recombinant protein were conducted using a confocal laser scanning microscope LSM 510 (Zeiss, Jena, Germany).

SDS-PAGE and Western Blot Analysis
The cell lysate was prepared by incubating cells with PBST (0.1% Triton-X 100 with PBS) containing a protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA) for 30 min on ice followed by sonication, and then, the lysate was mixed with a protein sample buffer and boiled. The protein samples were subjected to 12% SDS-PAGE and transferred to an NC membrane. The membranes were blocked in 5% milk in Tris-buffered saline containing 0.05% Tween 20 and were probed with each of the following antibodies: a GFP monoclonal antibody (Abm, Richmond, BC, Canada), PPV VP2 polyclonal antibody (Biorbyt, Cambridge, UK), CSFV-E2 monoclonal antibody (JENO Biotech, Chuncheon, GW, Republic of Korea), and His Tag monoclonal antibody (Abcam, Cambridge, UK). The membranes were then incubated with a horseradish peroxidase-coupled anti-mouse or rabbit IgG antibody (Cell signaling, Danvers, MA, USA), and the bound antibodies were detected using the enhanced chemiluminescence system (Merck Millipore, Burlington, MA, USA) according to the manufacturer's instructions.

Temperature and pH Inducible Cleavage
Bm5 cells infected with recombinant virus at 5 MOI were lysed on ice using Buffer B2 (20 mM Tris-HCl, pH 7.0 containing 500 mM NaCl, 0.1% Tween 20 and 1 mM EDTA). The cell lysate was treated at 25 • C for C-terminal cleavage of the Ssp DnaB mini-intein. The incubated samples were loaded on SDS-PAGE gels to confirm the cleavage activity of the Ssp DnaB mini-intein.

Effect of Mini-Intein Fusion Expression
The Ssp DnaB mini-intein is a powerful tool in protein engineering research because it can be self-cleaved by controlling the pH and temperature without the use of proteolytic enzymes. Effective purification of a target protein using the self-cleaving activity of the Ssp DnaB mini-intein has been extended to a wide range of applications [22][23][24], for example, purification of Ssp DnaB mini-intein fusion proteins via E. coli surface display [25] and purification using elastin-like polypeptide tags [26]. On the other hand, many types of useful proteins have been produced using the BES because it has several advantages over other expression systems. However, there have been no reports on the purification of BES-produced proteins using the SspDnaB mini-intein, probably because of the long time required for the mass production of target proteins in the BES. In general, intracellular self-cleavage is not a problem in the E. coli expression system because a sufficient amount of useful protein can be produced over a short time, even if expression is performed at pH 7.0 and 37 • C. Therefore, application of the Ssp DnaB mini-intein in the E. coli expression system was not difficult. However, the temperature and pH conditions required by insect cells are nearly identical to the conditions for self-cleavage of the Ssp DnaB mini-intein, and mass production of useful proteins using the BES generally requires more than 72 h. Therefore, in this study, use of the Ssp DnaB mini-intein in the BES was investigated with three different model proteins. Production of EGFP fused with the mini-intein (approximate 45 kDa) was observed at 36 h.p.i and increased with time ( Figure 2). Intracellular cleavage of the fusion protein occurred as expected at 72 h.p.i and was visualized by the detection of cleaved EGFP ( Figure 2B). The complete fusion protein was maintained for at least 60 h.p.i ( Figure 2C). Production of the mini-intein fusion protein was maximal at 72-84 h.p.i.
In addition, fusion with the mini-intein significantly enhanced the production of recombinant EGFP. This effect was clearly shown through a comparison of the fluorescence intensity ( Figure 3). The fluorescence intensity of the mini-intein fusion protein was increased by approximately 10 times. This result indicated that the Ssp DnaB mini-intein is sufficient for the purification of recombinant protein in the BES. Despite the production of fusion proteins under similar conditions as mini-intein cleavage, fusion expression of the mini-intein was useful in the BES because of the significantly enhanced production of recombinant proteins by the fusion with the mini-intein. The enhanced production of the fusion protein was enough to overcome the decreased production of the fusion protein due to intracellular self-cleavage. The increase in the expression level of foreign proteins through fusion with the Ssp DnaB mini-intein has been mentioned in the E. coli expression system [27], but not in the BES. To examine the optimal culture conditions for the production of the mini-intein fusion protein, the temperature and pH conditions for the culture of insect cells were investigated. To prevent intracellular self-cleavage of the fusion protein, recombinant virus-infected cells were cultured at various pH levels (from 5.8 to 7.0) and temperatures (from 24 to 30 • C). However, changing the cell culture conditions led to worse cell growth and eventually significantly decreased the production of the fusion protein ( Figure S1). Therefore, the normal conditions for insect cell culture were more suitable as the optimal conditions for the production of mini-intein fusion proteins.
Viruses 2018, 10, x 5 of 10 cleavage is not a problem in the E. coli expression system because a sufficient amount of useful protein can be produced over a short time, even if expression is performed at pH 7.0 and 37 °C. Therefore, application of the Ssp DnaB mini-intein in the E. coli expression system was not difficult. However, the temperature and pH conditions required by insect cells are nearly identical to the conditions for self-cleavage of the Ssp DnaB mini-intein, and mass production of useful proteins using the BES generally requires more than 72 h. Therefore, in this study, use of the Ssp DnaB mini-intein in the BES was investigated with three different model proteins. Production of EGFP fused with the miniintein (approximate 45 kDa) was observed at 36 h.p.i and increased with time ( Figure 2). Intracellular cleavage of the fusion protein occurred as expected at 72 h.p.i and was visualized by the detection of cleaved EGFP ( Figure 2B). The complete fusion protein was maintained for at least 60 h.p.i ( Figure  2C). Production of the mini-intein fusion protein was maximal at 72-84 h.p.i. In addition, fusion with the mini-intein significantly enhanced the production of recombinant EGFP. This effect was clearly shown through a comparison of the fluorescence intensity ( Figure 3). The fluorescence intensity of the mini-intein fusion protein was increased by approximately 10 times. This result indicated that the Ssp DnaB mini-intein is sufficient for the purification of recombinant protein in the BES. Despite the production of fusion proteins under similar conditions as mini-intein cleavage, fusion expression of the mini-intein was useful in the BES because of the significantly enhanced production of recombinant proteins by the fusion with the mini-intein. The enhanced production of the fusion protein was enough to overcome the decreased production of the fusion protein due to intracellular self-cleavage. The increase in the expression level of foreign proteins through fusion with the Ssp DnaB mini-intein has been mentioned in the E. coli expression system [27], but not in the BES. To examine the optimal culture conditions for the production of the miniintein fusion protein, the temperature and pH conditions for the culture of insect cells were investigated. To prevent intracellular self-cleavage of the fusion protein, recombinant virus-infected cells were cultured at various pH levels (from 5.8 to 7.0) and temperatures (from 24 to 30 °C). However, changing the cell culture conditions led to worse cell growth and eventually significantly decreased the production of the fusion protein ( Figure S1). Therefore, the normal conditions for insect cell culture were more suitable as the optimal conditions for the production of mini-intein fusion proteins.   The effects of the mini-intein on expression were further examined with two other proteins. The E2 and VP2 proteins are the main antigens of CSFV and PPV, respectively, and were expressed as fusions with the mini-intein. As with EGFP fusion, the production of both fusion proteins was observed from 36 h.p.i and maximum production occurred at a similar time, 72 h.p.i (Figure 4). Fusion with the mini-intein also greatly increased the production of both proteins. However, intracellular self-cleavage of both fusion proteins was observed much earlier, 48 h.p.i ( Figure 4B,E). This result indicated that the initiation time of intracellular self-cleavage varied depending on the target protein (Figures 2 and 4). Interestingly, expression of E2 non-fused with the mini-intein was not observed, but expression of E2 was significantly increased when fused with the mini-intein. Unsuccessful expression of recombinant E2 has been reported in similar studies [21,28].  The effects of the mini-intein on expression were further examined with two other proteins. The E2 and VP2 proteins are the main antigens of CSFV and PPV, respectively, and were expressed as fusions with the mini-intein. As with EGFP fusion, the production of both fusion proteins was observed from 36 h.p.i and maximum production occurred at a similar time, 72 h.p.i (Figure 4). Fusion with the mini-intein also greatly increased the production of both proteins. However, intracellular self-cleavage of both fusion proteins was observed much earlier, 48 h.p.i ( Figure 4B,E). This result indicated that the initiation time of intracellular self-cleavage varied depending on the target protein (Figures 2 and 4). Interestingly, expression of E2 non-fused with the mini-intein was not observed, but expression of E2 was significantly increased when fused with the mini-intein. Unsuccessful expression of recombinant E2 has been reported in similar studies [21,28].  The effects of the mini-intein on expression were further examined with two other proteins. The E2 and VP2 proteins are the main antigens of CSFV and PPV, respectively, and were expressed as fusions with the mini-intein. As with EGFP fusion, the production of both fusion proteins was observed from 36 h.p.i and maximum production occurred at a similar time, 72 h.p.i (Figure 4). Fusion with the mini-intein also greatly increased the production of both proteins. However, intracellular self-cleavage of both fusion proteins was observed much earlier, 48 h.p.i ( Figure 4B,E). This result indicated that the initiation time of intracellular self-cleavage varied depending on the target protein (Figures 2 and 4). Interestingly, expression of E2 non-fused with the mini-intein was not observed, but expression of E2 was significantly increased when fused with the mini-intein. Unsuccessful expression of recombinant E2 has been reported in similar studies [21,28].

Cleavage Activity of the Mini-Intein
To examine the cleavage activity of the mini-intein, the recombinant virus-infected cell lysate was collected at 72 h.p.i and treated at 25 • C and pH 7.0. Cleavage of the EGFP fusion protein began at 2 h and was completed after 12 h ( Figure 5A). Cleavage of the VP2 fusion protein occurred at 2 h and was completed after 4 h ( Figure 5C). However, production of the E2 fusion protein was reduced but cleaved E2 protein was not observed ( Figure 5B). These results suggested the mini-intein fused proteins were successfully cleaved under the proper temperature and pH conditions. Generally, pH 7.0 and 27 • C are suggested as the conditions for the self-cleavage of the Ssp DnaB mini-intein [13,29]. These conditions were also suitable for cleavage of the Ssp DnaB mini-intein fusion protein produced in the BES. The initiation and completion times of self-cleavage differed depending on the fused target proteins ( Figure 5). In our study, however, cleavage of E2 was observed as the intensity of the band corresponding to the fusion protein decreases with time but that the cleaved protein was extremely instable and was not detected ( Figure 5B). Poor production of E2 has been described in other reports [21].

Cleavage Activity of the Mini-Intein
To examine the cleavage activity of the mini-intein, the recombinant virus-infected cell lysate was collected at 72 h.p.i and treated at 25 °C and pH 7.0. Cleavage of the EGFP fusion protein began at 2 h and was completed after 12 h ( Figure 5A). Cleavage of the VP2 fusion protein occurred at 2 h and was completed after 4 h ( Figure 5C). However, production of the E2 fusion protein was reduced but cleaved E2 protein was not observed ( Figure 5B). These results suggested the mini-intein fused proteins were successfully cleaved under the proper temperature and pH conditions. Generally, pH 7.0 and 27 °C are suggested as the conditions for the self-cleavage of the Ssp DnaB mini-intein [13,29]. These conditions were also suitable for cleavage of the Ssp DnaB mini-intein fusion protein produced in the BES. The initiation and completion times of self-cleavage differed depending on the fused target proteins ( Figure 5). In our study, however, cleavage of E2 was observed as the intensity of the band corresponding to the fusion protein decreases with time but that the cleaved protein was extremely instable and was not detected ( Figure 5B). Poor production of E2 has been described in other reports [21].

Status of the Mini-Intein Fusion Protein
To investigate the reason for the enhanced production of the fusion protein, the intracellular status of the fusion protein was examined via immunofluorescence staining. Each recombinant protein except E2 was evenly dispersed in the cytoplasm when expressed in its non-fusion form ( Figure 6). However, the mini-intein fused recombinant protein was observed to aggregate in the cytoplasm ( Figure 6). The intensity of fluorescence of fusion proteins was also higher than that of non-fusion proteins. Aggregation might increase the stability of the protein against various proteases and may have resulted in the enhanced production of protein. Enhanced stability and production of foreign proteins by supramolecular assembly were reported in other studies [30][31][32][33]. Fusion expression of baculovirus polyhedrin [21,32] and human ferritin heavy chain [33] increased the stability and production of foreign proteins. In our study, the aggregation of foreign proteins did not affect the cleavage activity of the Ssp DnaB mini-intein and His tag affinity purification ( Figure S2). However, further study is required to establish whether the work can be successfully translated to a difficult to express protein because this work has been carried out with a model protein.
In conclusion, the Ssp DnaB mini-intein is a useful fusion partner in the baculovirus expression system for the easy purification and accumulation of target proteins.

Status of the Mini-Intein Fusion Protein
To investigate the reason for the enhanced production of the fusion protein, the intracellular status of the fusion protein was examined via immunofluorescence staining. Each recombinant protein except E2 was evenly dispersed in the cytoplasm when expressed in its non-fusion form ( Figure 6). However, the mini-intein fused recombinant protein was observed to aggregate in the cytoplasm ( Figure 6). The intensity of fluorescence of fusion proteins was also higher than that of non-fusion proteins. Aggregation might increase the stability of the protein against various proteases and may have resulted in the enhanced production of protein. Enhanced stability and production of foreign proteins by supramolecular assembly were reported in other studies [30][31][32][33]. Fusion expression of baculovirus polyhedrin [21,32] and human ferritin heavy chain [33] increased the stability and production of foreign proteins. In our study, the aggregation of foreign proteins did not affect the cleavage activity of the Ssp DnaB mini-intein and His tag affinity purification ( Figure S2). However, further study is required to establish whether the work can be successfully translated to a difficult to express protein because this work has been carried out with a model protein.
In conclusion, the Ssp DnaB mini-intein is a useful fusion partner in the baculovirus expression system for the easy purification and accumulation of target proteins.