Overexpression of soluble recombinant Thermus thermophilus ( Tth ) DNA polymerase in Escherichia coli BL21(DE3) using an MBP fusion tag as a solubility enhancer

Tth DNA polymerase is a thermostable enzyme derived from Thermus thermophilus and acts as a DNA polymerase and reverse transcriptase. Escherichia coli is used for large-scale enzyme production because of its cost-effectiveness, rapid growth, and increased recombinant protein expression, but inclusion bodies can be formed during intracellular protein expression, so the maltose-binding protein (MBP) tag was used to improve the expression of soluble protein. The Tth DNA polymerase gene was optimized to have a codon adaptive index of 1.00% and 60.64% Guanine and Cytosine (GC) content, then inserted into E. coli BL21(DE3), which harbors pD861-His- Tth DNA polymerase and pD861-MBP- Tth DNA polymerase. The induction and postinduction incubation time were optimized to express pD861-His- Tth DNA polymerase and pD861-MBP- Tth DNA polymerase in the soluble form. The total protein concentration of His- Tth DNA polymerase is 3.9095 mg/ml while for MBP- Tth DNA polymerase it is 33.541 mg/ml; protein levels after optimization based on densitometry analysis show MBP- Tth DNA polymerase is seven times higher than His- Tth DNA polymerase. This indicates that MBP tag fusion increases the amount of soluble protein produced.


INTRODUCTION
Tth DNA polymerase is a thermostable DNA polymerase enzyme derived from the thermophilic bacterium Thermus thermophilus, which exhibits dual activity as a DNA polymerase and reverse transcriptase so that it can be used in Reverse transcription-polymerase chain reaction (RT-PCR). It catalyzes the polymerization of DNA in the presence of Mg 2+ ions, thereby aiding the formation of cDNA from RNA (Auer and Landre, 1995;Moreno et al., 2005;Myers and Gelfand, 1991). Tth DNA polymerase has 5ʹ-3ʹ exonuclease activity but does not have 3ʹ-5ʹ exonuclease activity. Tth DNA polymerase has also been reported to have RNAse-H-like activity that plays a role in the degradation of DNA-RNA duplexes (Auer and Landre, 1995).
Recombinant protein technology is typically used for large-scale enzyme production (Tripathi, 2016). Among the many expression systems producing recombinant protein, Escherichia coli is commonly used due to its high density on an inexpensive substrate and relatively inexpensive cultivation costs. Moreover, its genetics has been well characterized, and there is a wide availability of mutant host strains (Sriwidodo et al., 2019). Common protocols for protein production in E. coli cells include cloning of target genes in expression plasmids, transformation in the host strain, and then induction of target protein synthesis (Chae et al., 2017). Protein production in E. coli can use two approaches, extracellular and intracellular Silaban et al., 2019;. Intracellular protein expression *Corresponding Author Iman Permana Maksum, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Sumedang, West Java, Indonesia E-mail: iman.permana @ unpad.ac.id occurs in the cytoplasm of E. coli, while extracellular expression will direct protein expression to the culture medium by adding signal peptides. The level of protein expression intracellularly has a higher rate than extracellularly (Choi and Lee, 2004;Su et al., 2006).
Various types of enzymes were successfully produced in the host E. coli, including the recombinant Tth DNA polymerase enzyme. The recombinant Tth DNA polymerase enzyme was successfully expressed in the host E. coli (Moreno et al., 2005). There have been previous studies on the recombinant DNA polymerase Tth enzyme successfully expressed in both T. thermophilus and E. coli hosts (Dabrowski and Kur, 1998;Moreno et al., 2005). In this study, protease-deficient strains, such as BL21, were used. BL21 is an E. coli strain lacking in ompT and lon, two proteases that can interfere with the isolation of intact recombinant proteins as an approach to increase target protein expression . In addition, E. coli BL21 is also able to live on media with limited nutrition. Therefore, with the use of the E. coli BL21 strain, protein recombinants can be expressed with low protease levels (Jeong et al., 2009).
Although E. coli expression systems are cost-effective and suitable for large-scale recombinant protein production, the lack of molecular chaperons and the reducing environment of the E. coli cytoplasm can lead to the formation of insoluble aggregates. Their formation is often undesirable because they generally lack biological activity (Gomes et al., 2016;Singh et al., 2015). The formation of inclusion bodies was observed in the expression of the recombinant Tth DNA polymerase enzyme in E. coli, so efforts are needed to increase the acquisition of the active enzyme in the cytoplasm (Moreno et al., 2005). To address this issue, a solubility enhancer is required to improve the yield of soluble recombinant proteins, such as maltose-binding protein (MBP). Previously, it was shown that MBP-tagged fusion protein is more abundantly expressed in the soluble fraction than His-tagged and GST-tagged fusion protein, indicating that MBP is a very efficient solubility enhancer for the soluble expression of recombinant protein (Raran-Kurussi and Waugh, 2012). For these reasons, the E. coli expression system and MBP were chosen for this work to express recombinant Tth DNA polymerase in E. coli BL21(DE3).
In this report, we evaluated the expression of recombinant Tth DNA polymerase in E. coli BL21(DE3). The Tth DNA polymerase gene was optimized using the preference codon of E. coli, then constructed on the expression plasmid. Escherichia coli BL21(DE3) was transformed with pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase plasmids. His-Tth DNA polymerase and MBP-Tth DNA polymerase were expressed as soluble forms in E. coli BL21(DE3). Therefore, it is highly expected that the production of the recombinant Tth DNA polymerase enzyme in the host E. coli BL21(DE3) is expected to be developed as a component of the RT-PCR kit for the detection of SARS-CoV-2.

Transformation of E. coli BL21(DE3)
Escherichia coli BL21(DE3) was grown overnight in Luria Betani (LB) media at 37°C before 1% of the culture was inoculated into 50 ml LB media containing 75 μg/ml kanamycin. Cells were grown until the OD 600 reached 0.4-0.5 and then placed in an ice bath for 30 minutes before centrifugation at 4,000 g for 10 minutes at 4°C. The cell pellet was resuspended in 25 ml of 0.1 M cold CaCl 2 and incubated for 60 minutes before centrifugation at 4,000 g for 10 minutes at 4°C. The cell pellet was resuspended in 1 ml of 0.1 M cold CaCl 2 . Escherichia coli BL21(DE3) was transformed with pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase by the heat shock method. The plasmid (1 µl) was added into a microtube containing 100 µl competent cells and incubated in an ice bath for 30 seconds and then placed in a water bath at 42°C for 45 seconds before cooling in an ice bath for 2 minutes. Subsequently, 900 µl of LB media was added to the microtube and incubated at 37°C for 1 hour. A 100 µl aliquot of transformed cells was spread on LB agar supplemented with 75 µg/ml kanamycin.

Optimization of induction time
Escherichia coli BL21(DE3) cells harboring pD861-His-Tth DNA Pol and pD861-MBP-Tth DNA Pol plasmids were grown overnight in LB media at 37°C. The starter culture was then transferred into three flasks containing 20 ml LB medium supplemented with 75 µg/ml kanamycin and incubated at 37°C with shaking at 200 rpm. Cells were grown until the OD 600 reached 0.4 (early log phase), 0.6 (mid-log phase), and 1.0 (late log phase). Protein expression was induced by the addition of 4 mM L-rhamnose and incubation at 37°C. Cells were harvested after 4 hours of incubation by centrifugation at 6,000 g for 20 minutes at 4°C. For each 1 g of pellet, a 4 ml bacterial protein extraction buffer containing 2 µl DNase and 2 µl lysozyme was added and the suspension was incubated for 25 minutes at room temperature before centrifugation at 15,000 g for 5 minutes at 4°C. The supernatant was collected as a soluble fraction, and 8 ml of solubilization buffer (8 M urea, 20 mM Tris-HCl, and 5 mM β-mercaptoethanol, pH 8.5) was added for each 1 g of pellet. The suspension was incubated at 100 rpm for 1 hour at room temperature and centrifuged at 12,000 g for 20 minutes at 4°C. The supernatant was collected as solubilized inclusion bodies and stored at −20°C for further analysis.

Optimization of postinduction incubation time
Escherichia coli BL21(DE3) cells harboring pD861-His-Tth DNA Pol and pD861-MBP-Tth DNA Pol plasmids were grown overnight in LB media at 37°C. The starter culture was inoculated into 50 ml LB media containing 75 μg/ml kanamycin. For His-Tth DNA polymerase, cells were grown until the OD 600 reached 0.4 and 1.0 for MBP-Tth DNA polymerase, and then 4 mM L-rhamnose was added to induce protein expression at 37°C.
After induction, cells were harvested at different times (4, 5, 6, 7, and 8 hours of incubation) by centrifugation at 6,000 g for 20 minutes at 4°C. Soluble protein and inclusion bodies were isolated following the previously described methods.

Overexpression of recombinant His-Tth DNA polymerase and MBP-Tth DNA polymerase under optimized conditions
Escherichia coli BL21(DE3) cells harboring pD861-His-Tth DNA Pol and pD861-MBP-Tth DNA Pol plasmids were grown overnight in LB media at 37°C. The starter culture was inoculated into a flask containing 50 ml LB media. For His-Tth DNA polymerase, cells were grown until the OD 600 reached 0.4 and then induced with 4 mM L-rhamnose at 37°C. Cells were harvested after 4 hours of incubation. For MBP-Tth DNA polymerase, cells were grown until OD 600 reached 1.0 and induced with 4 mM L-rhamnose at 37°C. Cells were harvested after 8 hours of incubation by centrifugation for 20 minutes at 6,000 g at 4°C. Protein extraction and inclusion bodies solubilization procedures were repeated to collect the soluble fraction and inclusion bodies.

Direct western blotting
Whole-cell extracts were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer's protocol (Bio-Rad). The membrane was immersed in 25 ml of blocking solution [5% nonfat dry milk in Tris Buffered Saline with Tween (TBST) (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% Tween 20)] and incubated for 15 minutes at room temperature. After incubation, the membrane was washed once with 10 ml of primary antibodies (penta-His antibody QIAGEN) for 10-20 minutes. The membrane was rinsed in 50 ml of wash buffer for 3 minutes before the addition of 10 ml of the substrate and incubated for 10-30 minutes.

Total protein quantification
The total protein yield was measured by the Bradford assay. A dilution series of standards were prepared using the Bovine Serum Albumin (BSA) solution (0.2-2 mg/ml). The Bradford reagent (1 ml) was added to 100 µl of the standards and samples, and the absorbance was measured at 595 nm after 10 minutes.

Construction of the recombinant plasmid
The Tth DNA polymerase synthetic gene was obtained from GeneBank (Accession No. AP008226.1), consisting of 2,502 nucleotides. Tth DNA polymerase gene shares approximately 50% similarity to the codon preference of E. coli. A high percentage of rare codons on the gene of interest will decrease recombinant protein expression (Gomes et al., 2016); therefore, the gene should be optimized to achieve a high codon adaptive index (CAI) and 40%-70% GC content (Parret et al., 2016;Silaban et al., 2019;. The Tth DNA polymerase gene was optimized using the OPTIMIZER software and the E. coli B preference codon (Puigbò et al., 2007) to achieve a CAI of 1.000% and 60.64% GC content. The optimized Tth DNA polymerase gene was inserted into the expression plasmids pD861-His and pD861-MBP, then synthesized by ATUM. pD861-His-Tth DNA polymerase contains the N-terminal His-tag, while pD861-MBP-Tth DNA polymerase contains the N-terminal MBP tag. The Histag was used as an affinity tag for purification purposes, and the MBP tag was used as a solubility enhancer. The plasmid map of pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase is shown in Figure 1.

Transformation of E. coli BL21(DE3)
The competent E. coli BL21(DE3) cells were chemically transformed using the heat shock method as it is a relatively inexpensive, simple method that does not require special equipment (Liu et al., 2014). Escherichia coli BL21(DE3) transformed cells were observed on LB agar containing kanamycin as a selection marker (Fig. 2). The plasmid contains gene encoding neomycin phosphotransferase II which is responsible for kanamycin resistance; therefore, the transformant cell harboring pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase will survive in the presence of kanamycin (Jang and Magnuson, 2013;Wegerer et al., 2008).

Optimization of the induction time
Induction time in different growth phases of E. coli plays a significant role in the production of soluble recombinant protein (Ahmad et al., 2018;Kaur et al., 2018). The results showed that His-Tth DNA polymerase and MBP-Tth DNA polymerase were expressed in soluble form when the induction was performed between the early exponential to late exponential growth phase (Fig. 3). To determine the optimal expression of the soluble fraction, semiquantitative densitometry analysis was carried out using ImageJ (Yosua et al., 2021), indicating that the highest yield of His-Tth DNA polymerase was achieved when the induction was carried out in the mid-log phase (Fig. 4a), whereas induction in the mid-log phase provided a high yield due to the rapid growth of E. coli leading to a higher rate of protein expression (Yosua et al., 2021). Meanwhile, the optimal induction of MBP-Tth DNA polymerase was in the late log phase (Fig. 4b), in line with a previous study that reported that the level of recombinant protein increased up to the late log phase (Noi and Chung, 2017).

Optimization of postinduction incubation time
To investigate the effect of postincubation time on Tth DNA polymerase expression, five different incubation times (4, 5,  Lanes 1, 3, and 5: soluble fraction of induced cells at different induction times (0.4, 0.6, and 1.0, respectively); Lanes 2, 4, and 6: inclusion bodies of induced cells at different induction times (0.4, 0.6, and 1.0, respectively). The red and black arrows correspond to His-Tth DNA polymerase and MBP-Tth DNA polymerase, respectively. 6, 7, and 8 hours) were evaluated, as a previous study suggested that postinduction incubation time can affect the recombinant protein yield (Fazaeli et al., 2019). The duration of postinduction incubation time is affected by several factors such as the strength of the promoter, concentration of inducer, solubility of recombinant protein, and intrinsic properties of the recombinant protein (Noi and Chung, 2017). The different incubation times after induction resulted in different yields of soluble protein (Fig. 5), with the highest expression of the His-Tth DNA polymerase reached within 4 hours of incubation, whereas MBP-Tth DNA polymerase reached maximal expression within 8 hours of incubation (Fig. 6).

Expression of recombinant His-Tth DNA polymerase and MBP-Tth DNA polymerase under optimized conditions
Recombinant His-Tth DNA polymerase and MBP-Tth DNA polymerase were successfully expressed as 95 and 135 kDa protein in E. coli, respectively. The expression of recombinant His-Tth DNA polymerase and MBP-Tth DNA polymerase was conducted under optimized induction and postinduction incubation time with the soluble fractions obtained before optimization used for comparison. As shown in Figure 7, His-Tth DNA polymerase was mainly expressed in the insoluble form when MBP-Tth DNA polymerase was expressed in soluble form, with the amount of both proteins increasing after optimization in Figure 8. Also, the  Lanes 1, 2, 4, 6, and 8: soluble fraction of induced cells at different incubation times (4, 5, 6, 7, and 8 hours, respectively); Lanes 3, 5, 7, and 9: inclusion bodies of induced cells at different incubation times (5, 6, 7, and 8 hours, respectively). The red and black arrows correspond to His-Tth DNA polymerase and MBP-Tth DNA polymerase, respectively. expression of MBP-Tth DNA polymerase produced more soluble protein than His-Tth DNA polymerase. This is in line with a previous study that reported that all MBP-tagged protein fusions yielded more soluble protein than His-tagged protein fusions (Raran-Kurussi and Waugh, 2012), as MBP serves as a "holdase" to maintain the passenger protein in a soluble form until it reaches its native conformation either by spontaneous folding or with the assistance of endogenous chaperones (Raran-Kurussi and Waugh, 2012). This mechanism may involve molecular interaction between MBP and hydrophobic amino acid residues existing on the incompletely folded passenger protein, thereby preventing aggregation (Guo et al., 2018;Raran-Kurussi and Waugh, 2012).

Western blotting
The expressions of pD861-His-Tth DNA polymerase and pD861-HMBP-Tth DNA polymerase were confirmed by Western blotting using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal loading control (Fig. 9).

Total protein quantification
The total protein concentration of the His-Tth DNA polymerase soluble fraction before and after optimization was 3.7002 and 3.9095 mg/ml, respectively. Meanwhile, the total protein concentration of the MBP-Tth DNA polymerase soluble fraction before and after optimization was 30.097 and 33.541 mg/ ml, respectively. These results indicate that the optimization process increased the total protein concentration. The concentration of total protein in the soluble fraction of MBP was eight times higher than the concentration of total protein in the soluble fraction of His.

CONCLUSION
In summary, soluble Tth DNA polymerase was expressed in E. coli BL21(DE3), with MBP-Tth DNA polymerase producing more soluble protein than His-Tth DNA polymerase due to its ability as a solubility enhancer.