Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

Autoinduction simplified the steps for protein production by omitting the need to monitor the correct time for addition of inducer. This method is preferable for industrially production of protein. By using lac operon-controlled expression system, it is possible to produce the protein without the addition of IPTG as inducer. In a conventional way, IPTG was added at the exponential growth phase. By using autoinduction, IPTG could be replaced by lactose in the lac operon-controlled expression systems. Lactose was already supplemented in the medium and it was more cost efficient than IPTG. Therefore, this study aims to employ autoinduction method for expression of our protein target as it is a more convenient way and it is suitable for industrial application. In this study, DNA polymerase from Geobacillus stearothermophilus (Bst DNA polymerase), an enzyme which generally used in loop-mediated isothermal amplification (LAMP) assay, was expressed using lac operon-controlled expression systems in Escherichia coli. Bst DNA polymerase was successfully expressed using autoinduction expression method in LB medium containing 0.2% Lactose.


Introduction
Detection of parasite and disease are generally carried out by RT-PCR.For example, detection of COVID-19, which was first discovered in China in 2019.Due to the massive spread of COVID-19, it is crucial to apply diagnostic tests that are fast and affordable to the broader community.Regardless, RT-PCR requires specialized equipment (e.g., PCR machines) and highly trained personnel and is relatively time-consuming.Alternative methods such as LAMP and RT-LAMP can be an answer because they allow more attainable detection methods without the need for a PCR machine, less time is required for detection, however sensitive and specific [1,2].The high specificity of LAMP method is due to the use of four to six primers and thereby, up to eight positions in DNA template can be recognized compared to only two positions in the PCR method [3,4].LAMP was first introduced by Notomi et al. [3] by utilizing DNA polymerase from Geobacillus stearothermophilus (Bst DNA polymerase) to amplify the target nucleic acids, instead of the typical DNA polymerase from Thermus Aquaticus that is used in PCR method [5].Despite the potential of Bst DNA polymerase, using commercially available enzyme is not economically feasible as they are still imported from abroad in Indonesia.Thus, it impacts the price, which is less affordable, hindering the utilization of these enzymes widely in Indonesia.The use of in-house production enzyme in Indonesia for routine use is highly desirable.
In previous investigation by Agustriana et al. [6], the construction of the gene encoding Bst DNA polymerase has been carried out.The gene was successfully cloned into the plasmid pD451-SR and transformed into E. coli BL21 star (DE3).Bst DNA polymerase was successfully purified to a high level of purity using the affinity chromatography method.The Bst DNA polymerase enzyme was confirmed by western blot.The pure enzyme Bst DNA polymerase has a specific activity of 813.56 U/mg.These results are in agreement with those reported by Rastgoo et al. [7] that the specific activity of DNA polymerase from Geobacillus sp.MKK from wild-type and mutant DNA polymerase is in the range of 700 to more than 900 U/mg indicating that Bst DNA polymerase in this research has the potential to be developed.
The most common way for producing enzymes is by conduction manual induction using Isopropyl βd-1-thiogalactopyranoside (IPTG) as inducer in the lac operon-controlled expression systems in Escherichia coli.Nevertheless, this method is less suitable for industrial application due to the complicated monitoring process for induction and the use of expensive inducer, IPTG [8][9][10].Studier (2005) developed the autoinduction method for synthetizing protein to overcome those problems.The operation of the lac operon regulatory elements in mixes of carbon source such as glucose, glycerol, and lactose under diauxic growth conditions is the basis for autoinduction and IPTG that was used as inducer was replaced by lactose [11].Highlevel expression of various proteins using autoinduction method were previously reported [12][13][14].In this study, large fragment Bst DNA polymerase from previous report by Agustriana et al. [6] was used and it was successfully expressed using autoinduction method.The yield of the enzyme and the bacterial biomass were investigated when several complex media were used and thereby can provide insight in the production of Bst DNA polymerase by utilizing autoinduction method in complex media.

Strain, vector and medium
For protein expression, E. coli BL21 Star TM (DE3) were utilized.The expression vector, pD451-SR (ATUM) containing T7 promoter, was utilized to create the plasmid pD451-SR_Bstpol, which included Bst DNA polymerase [6].Colonies carrying an expression vector were pre-cultured in Luria-Bertani medium (LB) with 0.4% glucose and 30 mg/L kanamycin.For culture, they were grown in LB enriched with 0.2% lactose and 30 mg/L kanamycin.

Transformation
The PEG technique, that was developed by Chung et al. [15], was used to transform the generated plasmid pD451-SR_Bstpol into E. coli BL21 star (DE3) with a number of modifications.At 37 °C for 16 hours, transformants were grown on LB-kanamycin agar plates.The growing colonies on the plate were then chosen for protein expression screening.

Expression of Bst DNA polymerase
E. coli BL21 Star TM (DE3) harboring the plasmid pD451-SR_Bstpol was grown in preculture medium and was incubated at 37 •C at 160 rpm.The pre-culture (1%, v/v) was then transferred into 50 mL of culture medium and was grown at 37 •C with agitation at 160 rpm for four days.A total of 5 mL cell culture was collected every 24 hours to examine the bacterial biomass at optical density of 600 nm and the yield of Bst DNA polymerase.The cell pellet was separated from the medium by centrifugation (8000 rpm, 6 min, 4 •C) and was resuspended by using Tris-HCl Buffer (25 mM, pH 8.0) supplemented with 1 mM PMSF.For protein extraction, the resuspended cell was sonicated 2 times on ice with amplitude of ultrasound probe at around 30%.Centrifugation (14,000 rpm, 4 •C, 15 mins) was conducted to separate the crude protein from the cell debris.

Determination of Bst DNA polymerase yield
According to the method developed by Laemmli [16], the crude protein at different incubation time was examined using SDS-PAGE on a 10% gel.By examining the target protein's band and comparing it to the linear equation generated from the BSA standard curve, the yield of Bst DNA polymerase was calculated.The ImageJ analysis program was used to complete this task [6].

Results and Discussion
Since the first report by Studier [11], expression of various proteins in E. coli using autoinduction methods has been continuously reported [12][13][14].Autoinduction method is better suited for high-throughput expression investigations since it eliminates the requirement to track the cultures' attenuation to choose the optimum time for induction.Additionally, autoinduction is a more economical way to produce protein, especially when scale-up fermentation is needed, given the expensive cost of IPTG [8][9][10].Glucose is preferred compared to other carbon sources during the initial growth phase and due to its role in catabolite repression, the protein is expressed in low-level [17].Catabolite suppression is lifted as glucose levels drop, which leads to the consumption of lactose and glycerol that is also supplemented in the autoinduction growth media.When lactose is utilized, ߚ-galactosidase engages in a reaction that converts lactose into allolactose and the lac operon is then physiologically induced by allolactose [18].Replacement of IPTG by inexpensive material such as lactose in the synthesis of protein in the T7 lac promoter-based expression system is preferrable for industrial application.
In this study, complex medium, LB is used as the autoinduction growth medium.In the typical LB medium, glucose is the sole carbon source.A total of 0.05% glucose was supplemented in LB medium.This concentration is enough to prevent the basal expression of recombinant protein in the early growth phase and subsequently leads to the growth of the E. coli [11].Meanwhile, a failure to block the expression of the recombinant protein due to not sufficient concentration of glucose at the early growth phase may limit the cell growth if the recombinant protein is highly toxic for the cell.To induce the synthesis of recombinant protein in the autoinduction method, the addition of lactose is needed.Based on the investigation by Studier [11], concentration of 0.2% of lactose is sufficient for induction of expression targeted protein.Thus, 0.2% lactose was added in the composition of LB to carry out autoinduction of Bst DNA polymerase.Among other complex media, LB was selected as the growth medium in this present study as it was reported as the common complex medium for expressing proteins in E. coli by autoinduction method by up to 60% [19].In this present study, large fragment Bst DNA polymerase that was constructed in the previous study was utilized [6].For the purpose of in-house production of Bst DNA polymerase in Indonesia, the enzyme was produced in the T7 lac promoter-based expression system in E. coli by autoinduction method.Incubation time is one of the crucial factors for synthetizing recombinant proteins.For this reason, investigation on the optimum incubation time for producing Bst DNA polymerase was performed.Bst DNA polymerase was incubated for 4 days (96 hours) and the yield of the enzymes was monitored every 24 hours.In the first attempt, the yield of Bst DNA polymerase was analyzed using SDS-PAGE.Secondly, the amount of the enzyme was calculated using imageJ software.As the standard for calculation, the known amount of BSA was also calculated in the similar approach as previously described.SDS-PAGE analysis of the known amount of BSA was shown in Fig. 1(A).The thickness of the band at each amount of BSA was examined as area under curved (AUC) by using imageJ software and the standard curve was shown as a graph in linear equation Fig. 1(B).In the linear regression model, R 2 was used as indication on the goodness of fit [20].Based on the standard curve presented on Fig. 1(B), R 2 value of about 0.9982 was examined indicating that the model is nearly perfect fit and thereby, it was used for further investigation on the yield of Bst DNA polymerase.Analysis of Bst DNA polymerase yield on the 10% gel of SDS-PAGE was presented in Fig. 2(A).Bst DNA polymerase was detected at molecular mass of about 68 kDa, which is in accordance with the previous report (Eva et al., 2022).After confirming that the enzyme was successfully expressed by autoinduction method, the yield of Bst DNA polymerase at each incubation time was investigated (Fig. 2(B)) and it showed that the highest yield (0.22 g/L culture) was obtained when the enzyme was incubated for 24 hours.Prolonging incubation time longer than 24 hours is not necessary as it caused the decreased on the yield of Bst DNA polymerase.Bacterial biomass at each incubation time was also measured (Fig. 3).After 24 hours, optical density of culture at 600 nm reached approximately 4.1 and the OD600 was detected at similar value after 96 hours incubation times.In contrast, the yield of the enzyme decreased steadily after 24 h incubation suggesting that the culture was likely already at stationary phase after 24 hours incubation.In the stationary phase, the nutrients for growth were limited leading to the death of the cell and subsequently, it caused the decreased amount of the recombinant protein at longer incubation time than 24 hours.

Conclusion
Bst DNA polymerase was successfully expressed in the T7 lac promoter-based expression system in E. coli by autoinduction method using LB medium supplemented with 0.2% lactose.The expression of the enzymes was found to be optimum after 24 hours incubation.

Figure 2 .
(A) SDS-PAGE analysis of the Bst DNA polymerase yield at different incubation time.Lane M: protein ladder; lane 1: soluble fraction after 24 hours incubation; lane 2: soluble fraction after 48 hours incubation; lane 3: soluble fraction after 72 hours incubation; lane 4: soluble fraction after 96 hours incubation.

Figure 3 .
Figure 3.The effect of incubation time on the bacterial biomass.