|Year : 2022 | Volume
| Issue : 3 | Page : 171-176
Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80
Sri Agung Fitri Kusuma1, Toto Subroto2, Ida Parwati3, Yaya Rukayadi4, Muhammad Fadhlillah2, Ruth Michellee Pardede1, Alif Virisy Berlian1, Gina Sabila1
1 Department of Biology Pharmacy, Faculty of Pharmacy, Padjadjaran University, Bandung, West Java, Indonesia
2 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University; Research Center of Molecular Biotechnology and Bioinformatics, Padjadjaran University, Bandung, West Java, Indonesia
3 Department of Clinical Pathology, Faculty of Medical, Padjadjaran University, Bandung, West Java; Department of Clinical Pathology, Dr. Hasan Sadikin General Hospital, Bandung, Indonesia
4 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Sedang, Malaysia
|Date of Submission||19-Jan-2022|
|Date of Decision||24-Apr-2022|
|Date of Acceptance||26-Apr-2022|
|Date of Web Publication||05-Jul-2022|
Dr. Sri Agung Fitri Kusuma
Department of Biology Pharmacy, Faculty of Pharmacy, Padjadjaran University
Source of Support: None, Conflict of Interest: None
pelB has been known as a successful signal peptide to translocate the protein target extracellularly in the Escherichia coli system. However, in our previous study, the yield of MPT64 protein extracellular recovery was still low and plenty of this protein was remain trapped in cytoplasm and periplasm. Recently, nonionic surfactants were efficiently reported to secrete recombinant protein extracellularly. Nonetheless, it must be clarified whether the surfactant supplementation can improve the yield of MPT64 extracellular protein significantly without giving impact on the structure of isolated MPT64 protein and can minimized the cell lysis effect. MPT64 protein secretion was carried out by comparing the effects of surfactants Tween 80 and Triton × 100 at various concentrations. Triton × 100 was able to increase the extracellular MPT64 protein gain up to 3 times higher than Tween 80 and it was in line with the greater level ratio of cell leakage of Triton × 100 compared to that of Tween 80. Similarly, the viable cell of the cultures decreased dramatically. However, both surfactants did not interfere the structure of MPT64 protein. In conclusion, Triton × 100 can be chosen as the supporting surfactant to assist the act of peptide signal in improving the resulting of MPT64 extracellular protein.
Keywords: Extracellular, MPT64, pelB, Triton × 100, Tween 80
|How to cite this article:|
Kusuma SA, Subroto T, Parwati I, Rukayadi Y, Fadhlillah M, Pardede RM, Berlian AV, Sabila G. Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80. J Adv Pharm Technol Res 2022;13:171-6
|How to cite this URL:|
Kusuma SA, Subroto T, Parwati I, Rukayadi Y, Fadhlillah M, Pardede RM, Berlian AV, Sabila G. Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80. J Adv Pharm Technol Res [serial online] 2022 [cited 2022 Aug 14];13:171-6. Available from: https://www.japtr.org/text.asp?2022/13/3/171/349835
| Introduction|| |
The strategy to evade the host's immune system is an important virulence factor of Mycobacterium tuberculosis in maintaining the multiplication of this bacterium in infected host cells. Several M. tuberculosis secreted proteins facilitate this bacterium by regulating antiapoptotic and proapoptotic molecules to inhibit the apoptosis of infected macrophages., MPT64 is one of the proteins secreted from M. tuberculosis with antiapoptosis mechanism on RAW264.7 macrophages by upregulating the Bcl-2, miRNA21, and nuclear factor-kappa B (NF-κB). The most interesting of this protein is its specification to distinguish the M. tuberculosis complex from other mycobacteria bacterial species. This protein has great potential for M. tuberculosis identification using immune chromatographic methods through the antigen–antibody binding process., Hence, this protein must be present in adequate concentration to generate antibodies against MPT64 to be constructed in immunochromatographic diagnostic kit. However, MPT64 is difficult to be conventionally isolated from the original source due to the abundant of other secreted tuberculosis proteins which difficult to purify, and it is grouped as high-risk air infect pathogen.
In our previous study, this protein was produced by expressing the synthetic gene coded for the MPT64 protein in Escherichia coli BL21 (DE3) and secreted extracellularly by constructing the gene fused with pelB signal peptide., We found that the pelB success to translocate the MPT64 extracellularly, but the yield was still low and plenty of this protein was remain trapped in cytoplasm and periplasm. Thus, to enhance the secretory potential, the growth medium of E. coli BL21 (DE3) was augmented with surfactants, which allows the production of highly efficient extracellular target proteins in E. coli but does not impair the activity of the target protein., Triton × 100 and Tween 80 were reported effectively secreted protein targets in high levels compared to other surfactants such as CaCl2, sodium dodecyl sulfate (SDS), and glycine in E. coli. Therefore, in this study, the optimized conditions related to the secretion process of MPT64 protein were conducted and the extracellular MPT64 structure was evaluated to validate the structural integrity of the protein required to maintain antigen epitopes for antibody production.
| Materials and Methods|| |
The materials used are E. coli BL21 (DE3) transformant, Luria Bertani (LB) (Himedia), L-rhamnose (Sigma-Aldrich), Tween 80 (Sigma-Aldrich), Triton × 100 (Sigma-Aldrich), and SDS PAGE components.
Optimization of recombinant MPT64 protein secretion
Optimization of MPT64 protein secretion was carried out by comparing the effects of surfactants Tween 80 and Triton × 100 at various concentrations as follows: 0%, 0.1%, 0.2%, and 0.5%. A total of 6 mL of bacterial starter were inoculated into four Erlenmeyer flasks containing 294 mL of liquid LB, then 300 L of kanamycin (100 g/mL) were added to each of the Erlenmeyer flasks and incubated at 37°C, 180 rpm, optical density OD600 (0.6–0.8). After that, the bacterial culture was measured for its OD at a wavelength of 600 nm (t0) and 10 mL of the bacterial culture was taken and centrifuged at 6000 g at 4°C for 20 min. The cell culture was then induced with the addition of Rhamnose to a concentration of 4 mM, then incubated again at 37°C, 180 rpm for 24 h. After 24 h, the OD600 of bacterial culture was remeasured (t24) and 10 mL of cell culture was taken for centrifugation at a speed of 6000 g at 4°C for 20 min. Then, the surfactant was added to each Erlenmeyer flask with each concentration of 0, 0.1, 0.2, and 0.5%, and incubated at 37°C, 100 rpm for 6 h. After that, the bacterial culture OD600 was measured again (t30) and 10 mL of cell culture was taken and centrifuged at 6000 g at 4°C for 20 min. Before the isolation of the media fraction, phenylmethylsulfonyl fluoride was added to each culture until the final concentration was 0.1 mM. A total of 10 mL of the culture taken at t0, t24, and t30 were centrifuged to separate the supernatant from the pellets. Proteins in the media fraction were characterized using the SDS-PAGE method and quantified using ImageJ software.
Effects of surfactants
Ten milliliters of overproduced cultures at t0, t24, and t30 were centrifuged at 10,000 g, 4°C, for 5 min. Then, its absorbance at OD260 was checked to measure the DNA concentration in the transformant cultures. The higher the absorbance, the higher the concentration of DNA in the sample, which indicates the number of cells undergoing lysis. As confirmation of cell viability, each sample at t0, t24, and t30 was inoculated as much as 10 L on the surface of solid LB medium containing kanamycin. The culture on solid media was then incubated at 37°C for 24 h and the number of colonies observed was counted.
| Results and Discussion|| |
In our previous study, the optimal conditions for MPT64 gene expression have been conducted, but the acquisition of extracellular MPT64 protein as a protein target was still low. In line with this, it has been widely reported that the challenge to express extracellular proteins in E. coli is always distributed in the periplasm. To overcome this drawback, the acquisition of extracellular MPT64 protein can be increased by improving the leakage of specific host cell membranes by constructing the cell host into a leaky phenotype. Such phenotypes can be induced through several ways, as follows: mutation or deletion of membrane components, addition of permeability enhancers (glycine, calcium, Triton X-100, etc.), or coexpression of proteins with lytic activity.,,,,
Triton × 100 and Tween 80 are the most widely used nonionic surfactants to increase the permeability of living cell membranes.,,,, Therefore, this evidence based bring this study to investigate the effect of both surfactants in improving the yield of MPT64 extracellular recovery. As seen in [Figure 1] and [Figure 2], the thickness of the protein band in each cell fraction described the different protein concentrations at various surfactant concentrations. The protein concentrations were demonstrated in [Table 1] and [Table 2] and the different effects of each surfactant in various concentrations on the MPT64 extracellular protein recovery can be described clearly in [Figure 3] and [Figure 4]. The supplementation of Triton × 100 (0.5% v/v) into the growth medium could increase the extracellular MPT64 protein gain up to 3 times higher than using Tween 80 at the same concentration. The different effects of both surfactants on membrane permeability were predicted by the fact that nonionic surfactants do not dissociate when dissolved in water and have the widest range of properties depending on the hydrophilic-lipophilic equilibrium ratio (HLB). If the HLB value of the surfactant is higher than 10, then the nonionic surfactant tends to be more hydrophilic. As a result, the surfactant layer lowers the surface tension of the water more than the surface tension of the oil. Surfactants with low HLB values are more soluble in oil (lipophilic), while surfactants with higher HLB values are more soluble in water (hydrophilic). This supports the evidence that Triton × 100 (HLB = 13.5) with a lower HLB value than Tween 80 (HLB = 15.0) is able to interact more strongly with bacterial cell membranes composed of phospholipids. In addition, we must confirm the effect of the surfactants to the MPT64 structure. From [Figure 5], we can see that the antibody of the kit diagnostics can capture the MPT64 successfully. It is indicated that both surfactants did not interfere the MPT64 structure. Thus, the overproduction of MPT64 protein can be combined with Triton × 100 surfactant at the late stage of incubation.
|Figure 1: Effect of Tween 80 on MPT64 protein gain in different fractions (a) medium (b) periplasm (c) cytoplasm|
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|Figure 2: Effect of Triton × 100 on MPT64 protein gain in different fractions (a) medium (b) periplasm (c) cytoplasm|
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|Figure 3: Distribution of MPT64 production after Triton × 100 treatment in different concentrations|
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|Figure 4: Distribution of MPT64 production after Tween 80 treatment in different concentrations|
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|Figure 5: The identification of secreted MPT64 protein influenced by (A) Triton-×100; (B) Tween 80 (a) cytoplasm (b) periplasm, (c) medium|
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However, the concentration used must be considered because several studies have reported the occurrence of cell death after prolonged exposure to these surfactants.,, From this study, both surfactants were shown to be able to release higher extracellular MPT64 protein compared to untreated cells, presented in [Table 3]. The level ratio of cell leakage between Triton × 100 (0.5%) and Tween 80 (0.5%) against the control cell was 86.07% and 66.35%, respectively. From [Table 4], at the 30th h after the addition of surfactant, the culture added with Triton × 100 experienced a large decrease number of viable cells for about 8%–20% at 24 h. Therefore, we hypothesized that both surfactants could interact with the cell membrane, but the high level of MPT64 protein is still trapped in the cytoplasm and periplasm. We predicted that this was due to the ineffectiveness of the pelB signal peptide used in translocating MPT64 protein from the cytoplasm to the periplasm, so that the acquisition of MPT64 extracellular protein is still not optimal. In the future, it is possible to optimize the use of signal peptides to increase the acquisition of extracellular MPT64 protein with the support of surfactants to release the accumulated MPT64 protein from periplasm to medium.
| Conclusion|| |
This study showed the efficiency of using Triton × 100 in supporting the action of the pelB signal peptide in translocating the extracellular MPT64 protein.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]