Cloning, expression, and purification of fusion antigens MPT83 and ESAT6 from the local strain of Mycobacterium tuberculosis in Escherichia coli as a seed vaccine candidate against tuberculosis

Tuberculosis (TB) is an infectious disease caused by M. tuberculosis that can affect multiple organs of the body. It mostly attacks the lungs, termed pulmonary TB, and can spread to other organs, also known as extrapulmonary TB. People with pulmonary TB can infect others by coughing up the bacterium [1]. M. tuberculosis is resistant to acidity due to the high lipid content of its cell membrane [2, 3]. Additionally, susceptibility to ultraviolet (UV) light and radiation allows the bacteria to spread more easily at night [4-6].

About 10.6 million people fell ill with TB in 2022, with 1.3 million of them dying, including those with HIV [1]. Indonesia contributes 10% of TB incidents globally, ranking second after India [7].  The death toll in Indonesia due to TB reached more than 144,000, which is higher than in previous years [8]. Eighty percent of Indonesia's TB patients are adults of employment age, which has devastating social and economic consequences [8]. The conditions favor TB bacteria to thrive, contributing to the disease's widespread distribution. Coinfection of TB/HIV, which requires a complex combination of therapies and often causes drug-drug interaction, and the emergence of multidrug-resistant TB only adds fuel to the fire [9]. To overcome this issue, developing cost-effective vaccines and rapid as well as rapid and accurate diagnostic technologies is essential. TB infection occurs when individuals inhale droplet nuclei carrying TB germs [10], leading to four possible outcomes: a negative tuberculin skin test, active TB, latent TB, or reactivation of latent TB into active TB within months to years [11].

Diagnostic tests and immunizations based on MPT83 (Rv2873) and Rv3875 have potential [12,13]. M. tuberculosis complex (MTC) species have these proteins. The M. tuberculosis Region of Difference 2 (RD2) region contains the Rv2873 gene and the Rv3875 gene, which codes for MPT83 and a 6-kilo Dalton secretory protein bearing the ESAT6 antigen, respectively. ESAT6 antigen has been reported specific to healthy and dividing cells and not found in most other mycobacterium types including M. bovis BCG. Several studies confirm that MPT83 and ESAT6 elicit a strong hypersensitivity reaction, and increase interferon release in TB patients, thus potentially enhancing the immune response against TB [14,15]. Both MPT83 and ESAT6 antigens serve as virulence markers in M. tuberculosis strains. They have been found only in complex M. tuberculosis strains and could be used in T-cell-based TB vaccines and diagnostics [16-18]. In addition, ESAT-6 has the ability to drive and stimulate protective immune responses, protecting against long-term chronic infections or post-exposure, and can induce immunity against M. tuberculosis pathogens, thus having great potential for use in TB vaccine development [19]. Previous studies have shown that mice treated with MPT83 homologous monoclonal antibodies exhibited improved outcomes when infected with M. tuberculosis [20]. Therefore, both these antigens are appropriate highly immunogenic TB seed vaccine candidates as well as for T-cell-based TB diagnostic tests.

In this study, a fusion antigen protein made of the MPT83 and ESAT6 genes through recombinant DNA technology was developed. To produce the fusion antigen protein, we constructed the fusion plasmid pGEM-T Easy-Rv2873 plus Rv3875, by inserting the Rv3875 gene encoding the ESAT6 protein into the pGEM-T Easy-Rv2873 vector at the BamHI/HindIII cloning site [21]. Subsequently, the fusion genes for Rv2873 and Rv3875 were subcloned into the NheI/HindIII cloning site of the expression vector pTrcHisA to create pTrcHisA-Rv2873 + Rv3875. Recombinant 6XHis-tagged fusion protein MPT83 plus ESAT6 was produced by expressing these genes in the Escherichia coli (E. coli) BL-21strain, making it a potential addition to improved TB vaccines. By successfully expressing this fusion protein as a seed vaccine candidate, we are at a promising initial stage in the development of an effective TB vaccine. This progress paves the way for further research to evaluate the ability of this fusion protein to stimulate a strong and protective immune response, thereby contributing significantly to global efforts to control and eliminate TB.

Materials

JM109 and BL-21 E. coli cells were purchased from Promega, USA. pTrcHisA vector was purchased from Thermo Ficher Scientific. BamHI and HindIII restriction enzymes were purchased from NEB New England Biolabs.  PCR kits with Master Mix GoTaq green were purchased from Promega, USA. Ni⁺²-NTA affinity chromatography was purchased from GoldBio St. Louis, Missouri. LB media, Ampicillin, IPTG, X-gal, imidazole, PBS buffer, and Tris-HCl were purchased from Sigma.

DNA of M. tuberculosis sample preparation

M. tuberculosis isolate was obtained from the culture collection of Wahidin Sudirohusodo Hospital Makassar, Indonesia [22]. Any procedure dealing with human subjects was conducted in compliance with ethical guidelines and had been approved by the Ethics Committee at the Faculty of Medicine Hasanuddin University, Makassar, Indonesia, with an ethics approval number. 24/UN4.6.4.5.31/PP36/2024. The DNA of M. tuberculosis was extracted using the method described previously [22]. DNA extract was stored at -20 ^oC until used in PCR.

Primer design and Rv3875 gene amplification

Rv3875 gene of extracted M. tuberculosis DNA was amplified by PCR with Master Mix GoTaq green (Promega, USA, Cat No. M7122), and primers: Rv3875-F (5’-gcgggatcc atgacagagcagcagtgg-3’) and Rv3875-R (5’-ccgaagcttctatgcgaacatcccagtg -3’), which were designed to introduce BamHI restriction sites before the start codon and HindIII restriction sites after the end codon, respectively. Denaturation at 94 ^oC for 30 seconds, annealing at 55 ^oC for 30 seconds, and extension at 72 ^oC for 30 seconds was repeated 30 times after a 5-minute pre-denaturation at 90 ^oC. The longest period of extension occurred at 72 ^oC for seven minutes. The PCR result was then run electrophoresis on 1.0% agarose gel [21, 22].

Purification of PCR products

The PCR products of the Rv3875 gene were purified using an ez-10 column in the Geneaid kit (Qiagen). Reagents for DNA binding, washing, and elusion were included in the kit, all of which were applied in the purification procedure. The main objective of PCR product purification was to acquire contaminant-free DNA fragments appropriate for ligation into the pGEM-T vector. The agarose gel was sliced and homogenized, and then the PCR findings were placed in Eppendorf tubes with around 50 microliters of PB buffer solution to begin the purification process. The supernatant was obtained by centrifuging the remaining mixture at 13,000 rotations per minute for two minutes at ambient temperature. Following the removal of the supernatant, a volume of 700 microliters of PEP washing buffer was introduced into the spin column. Afterward, the sample was spun at a speed of 13,000 rpm for two minutes as an additional wash step for the PCR products. In the last step, 35 microliters of Elution buffer were added to the spin column and centrifuged again to collect the eluate. Electrophoresis was performed on a 1% agarose gel to verify the high purity level.

ESAT6 protein-encoding Rv3875 gene ligation into pGEM-T easy-Rv2873 plasmid

After properly mixing 50 μL of E. coli JM109 competent cells with 10 μL of the ligation product that had been inserted Rv3875 gene, the final volume was 100 μL. To serve as positive control, we cultivated competent E. coli JM109 cells with and without antibiotics (ampicillin, 100 μg/mL). The ligation product and E. coli JM109 competent cells were placed in three separate tubes and stored at 4 ^oC for 30 minutes. The samples were then exposed to a heat shock at 42^oC as described previously [23]. After that, the culture tubes spent 3 hours in a shaker incubator set at 37 ^oC and 150 rotations per minute. The samples were centrifuged for two minutes at 13,000 rpm. The vacuum centrifuged ligation products reached 100 microliters. After adding 0.8 mg 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal), ampicillin (100 μg/mL), and Isopropyl β-D-1-thiogalactopyranoside (IPTG) (40 micromolar), the cells were grown at 37°C for 15-18 hours.

Sequencing of the recombinant plasmid pGEM-T Easy- Rv2873 +Rv3875

DNA sequencing was performed to validate the nucleotide sequence. Bigdye Terminator sequencing was utilized on ABI PRISM 310 Biosystem hardware on this recombinant plasmid, pGEM-T Easy- Rv2873 +Rv3875. The sequencing results were visualized and evaluated by using Bioedit v.7.0.10.

Expression of recombinant protein fusion of MPT83 and ESAT6

To make the recombinant plasmid pTrcHisA-Rv2873+Rv3875, the Rv2873 and Rv3875 genes were fused to produce a fusion protein and then introduced into the pTrcHisA expression vector [24]. In order to produce recombinant fusion proteins consisting of MPT83 and ESAT6, the pTrcHisA-Rv2873+Rv3875 plasmid was introduced into Escherichia coli BL-21cells through transformation methods. The transformed cells were incubated overnight in a shaker incubator maintained at a temperature of 37 °C. After incubation, 4 mL of the bacterial recombinant culture was utilized as a sample without IPTG (MPT83 + ESAT6 non-induction, -IPTG), and the rest of the culture was diluted and added to fresh Luria-Bertani media. To reduce the amount of inclusion body proteins and maximize the production of MPT83 and ESAT6 proteins, 40 micromolars of IPTG was added to the final 5 mL of bacterial recombinant culture, and the mixture was incubated at 16 ^oC for 6-7 h until optical density (OD) of the cell cultures about 2.0-4.0.

After incubation, the E. coli BL-21 cultured cells were harvested by 13,000 rpm centrifugation for five minutes at 4 °C. The pellet was next mixed with a nutrient-rich 1X phosphate-buffered saline (PBS) solution containing 1% Triton X-100 with pH 7.5. Sonication was performed intermittently for 30 seconds using a 20 kHz frequency to disrupt the bacterial cell walls, allowing for the release of biomolecules from the E. coli BL-21 strain cells into the clear liquid [20]. To isolate this recombinant protein from the bacterial cellular debris, the material had to be centrifuged at 13,000 rpm for a minute while keeping the temperature at approximately 4°C.

Protein expression was assessed through the utilization of 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [16, 25], employing the recombinant proteins MPT83 and ESAT6 as reference standards in comparison to a conventional protein ladder (Tiagen, Biotech Beijing).

Purification of recombinant protein fusion of MPT83 and ESAT6

Recombinant protein fusion of MPT83 and ESAT6 was purified using nickel-nitrilotriacetic acid (Ni²⁺-NTA) affinity chromatography (GoldBio St. Louis, Missouri) with a His Tag-agarose matrix. Following column washing, this recombinant protein was eluted using elution buffer 0.5 M Tris-HCl buffer containing imidazole in varied concentrations (100-300 mM). SDS-PAGE gel electrophoresis was performed to evaluate the purified recombinant protein, which was then stained with Coomassie Brilliant Blue from Merck [25].

Amplification of ESAT6-encoding Rv3875 gene

The amplification of the Rv3875 gene was successfully achieved and showed a band of 288 bp (Figure 1A). The PCR product size matched that reported by GenBank (Access Number: KJ095583 for Gene ID: 886657), indicating that the Rv3875 gene, which includes the ATG start codon, is 288 bps in length and codes for the ESAT6 protein. In the absence of DNA, the negative PCR control showed no bands. DNA purification was successful because, as shown in Figure 1B, agarose gel electrophoresis revealed the presence of a 288 bps DNA band free of dimers and dimerization products.

Constructing the Rv3875 gene in the pGEM-T Easy-Rv2873 subcloning plasmid and transformation

In this study, the Rv3875 gene was cloned for further investigation and analysis. The pGEM-T Easy- Rv2873 +Rv3875 vector was constructed by inserting the target gene (gene of interest) into the pGEM-T Easy- Rv2873 cloning plasmid. We determined that the 660-bp long Rv2873 gene encodes the MPT83 protein and that the 288-bp long Rv3875 gene encodes the ESAT6 protein.

E. coli recombinant white colony transformation and screening results are shown in Figure 2. As depicted in Figure 2A, the absence of bacterial colonies was observed on Luria-Bertani agar plates when utilized as a negative control, specifically with competent E. coli JM109 cells lacking plasmids. This observation indicated no growth of E. coli JM109 cells without plasmids either autonomously or due to contamination. Figure 2B shows the blue colony which carries the vector pGEM-T Easy as transformation control. The experimental procedure included the introduction of pGEM-T Easy- Rv2873 into E. coli JM109 competent cells are depicted in Figure 2C. A collective sum of 396 bacterial colonies was seen on the Luria-Bertani plates. The utilization of the pGEM-T Easy- Rv2873 vector for transfection has yielded positive outcomes. The E. coli JM109 competent cells were subjected to transfection using the recombinant vector pGEM-T-Easy-Mpt83+Rv3875, as depicted in Figure 2D. A transformation effectiveness of 62.9% is inferred from the detection of 249 white bacterial colonies. Transformation of the recombinant plasmid encoding Rv3875 has been successful, as evidenced by the appearance of these white colonies.

Isolation and characterization of the recombinant plasmid pGEM-T Easy-Rv2873 +Rv3875

The recombinant vector pGEM-T Easy-Rv2873+Rv3875 exhibited DNA fragments of 288 bp (from Rv3875 gen alone) and 3678 bp (from the pGEM-T Easy- Rv2873) upon restriction with BamHI and HindIII enzymes, respectively (Figure 3). The pGEM-T Easy- Rv2873 vector has a length of 3678 base pairs, while the Rv3875 gene that has been inserted into it has 288 base pairs in length. The construction of the recombinant vector, pGEM-T Easy- Rv2873+Rv3875, was achieved through the insertion of the DNA of the Rv3875 gene into the pGEM-T Easy- Rv2873 vector, as seen in Figure 3.

White E. coli colonies harboring the MPT83 and ESAT6 fusion protein-encoding Rv2873 and Rv3875 genes were identified using the PCR colony technique (Figure 2C). The PCR result showed a single DNA band of 1,371 bps. Insert DNA from white colonies harboring the Rv2873 and Rv3875 genes measures 948 bps (columns 1-2) in Figure 4A. However, neither the Rv2873 nor the Rv3875 DNA inserts were found (Figure 4A, column 3).

Re-inoculation was conducted on a solid Luria-Bertani medium supplemented with 100 micrograms/mL of ampicillin subsequent to the identification of the converted white and isolated colony. Four colonies with white pigmentation were cultured in liquid Luria-Bertani media, supplemented with 100 micrograms/mL of ampicillin. The vectors were subsequently isolated from these colonies through the utilization of the QIAprep Spin Miniprep Kit manufactured by Qiagen, USA. The extraction resulted in the successful isolation of the recombinant vector pGEM-T Easy- Rv2873 +Rv3875, as confirmed by inspecting and displaying the electrophoresis results on a 1.5 % agarose gel (Figure 4B). Overall, the study demonstrated the successful isolation of the recombinant vector containing the Rv2873 and Rv3875 genes, which encode the MPT83 and ESAT6 fusion proteins, respectively, through the PCR colony method and subsequent vector extraction using the QIAprep Spin Miniprep Kit. The electrophoresis results confirmed the desired recombinant fusion plasmids in the three white colonies measure 948 bps (columns 1-3) in Figure 4B.

Recombinant plasmid sequencing

As depicted in Figure 5, the sequencing results showed conformity with the combined nucleotide sequences of the Rv2873 and Rv3875 genes encoding MPT83 and ESAT6 proteins, respectively. Combining these two genes produces a band of 948 bps, which begins with the start codon ATG and stop codon TGA. This sequence encodes 315 amino acids from the methionine (M) residue to the leucine (L). These sequencing results confirmed that fusion plasmid pGEM-T Easy- Rv2873 + Rv3875 was successfully created, which also provided crucial information regarding the encoded MPT83 and ESAT6 proteins.

Transformation and culture of E. coli Strain BL21(DE3) cells with pTrcHisA-Rv2873 +Rv3875 to generate fusion MPT83 Plus ESAT6 recombinant protein

Introducing the plasmid pTrcHisA-Rv2873 +Rv3875 into E. coli strain BL-21 cells yielded the results in Figure 6A; the bacteria multiplied and formed white colonies on the Agar Luria-Bertani culture. Subcloning is complete when white colonies form, proving that the fusion genes Rv2873 and Rv3875, which code for the MPT83 and ESAT6 proteins, respectively, have been achieved through the insertion into the pTrcHisA vector. Errors in expression in the lacZ gene of the pTrcHisA vector generated the white coloration of the E. coli colonies.

The E. coli strain BL-21 containing this recombinant fusion protein was then cultured in liquid LB medium with and without 40 micromolar IPTG induction for six hours (Figure 6B), and then the pellets were collected. The results were then analyzed on SDS-PAGE, as shown in Figure 6C. The cultivation of the cells with the pTrcHisA-Rv2873+Rv3875 plasmid by induction with 40 micromolar IPTG resulted in a significant protein band with molecular weight 28 kDa (Figure 6C column 1 and 3), but not found in the absence of IPTG (Figure 6C column 2 and 4). This result confirmed that MPT83 and ESAT6 are soluble proteins presented inside the cells. 

Figure 7 shows the purified recombinant MPT83-ESAT6 fusion protein. Figure 7 (lanes 1-2) compares cells without and with IPTG, similar to Figure 6C. The next lanes show the success of purifying the MPT83 plus ESAT6 fusion protein, demonstrating a single protein band of 28 kDa of molecular weight.

In this study, we produced MPT83 and ESAT6 fusion antigen proteins by cloning the Rv3875 gene encoding the ESAT6 protein into the previously created pGEM-T Easy-Rv2873 vector [21] to produce the pGEM-T Easy- Rv2873 + Rv3875 fusion plasmid. Similar to previous research, we determined that the 660-bp long Rv2873 gene encodes the MPT83 protein [14] and the 288-bp long Rv3875 gene encodes the ESAT6 protein. Overhanging bases (T) can be found at both ends of the linear pGEM-T Easy- Rv2873 vector, facilitating a more efficient PCR product ligation procedure by preventing self-ligation at the target insertion site with T-overhangs (X). To enhance the ligation process, a larger ratio of inserts to vectors—three to one—was employed. At thirty degrees Celsius, T4 DNA ligase is at its most active.

The Rv3875 gene ligation product and the pGEM-T Easy-Rv2873 cloning vector were then transformed into E. coli JM109 competent cells and the subsequent blue-white screening were selected because it has the ampicillin resistance (Amp^r) site and the lacZ gene, as reported in previous studies [24, 26]. To introduce recombinant DNA into E. coli cells, scientists used a thermal shock technique that briefly opened the cell membrane [26]. The rapid inflation and deflation of the cell wall brought about by the alternating cold and hot shocks allowed outside plasmid DNA to pass through the cell wall and enter the cell.

During the transformation and screening process of E. coli recombinants, different outcomes were observed for the transformed cells. Competent cells of E. coli strain JM109 that did not carry the plasmid could not grow. As a null hypothesis, this finding verifies that the presence of the altered plasmids is responsible for the growth seen in the other samples. E. coli JM109 containing the pGEM-T Easy plasmid without genes were grown with blue colony (Figure 2B). Whereas the presence of white colonies harboring the vector pGEM-T Easy- Rv2873 (Figure 2C) and pGEM-T Easy-Rv2873+Rv3875 recombinant plasmid (Figure 2D) was confirmed. The white colonies demonstrated that the corresponding vector had successfully transformed and was present in the E. coli JM 109 competent cells. These findings support the successful cloning and transformation of the pGEM-T Easy- Rv2873 vector and the recombinant plasmid pGEM-T Easy-Rv2873 +Rv3875 into E. coli JM109 competent cells.

Blue-white screening identified bacterial cells that successfully contained recombinant plasmids with gene inserts. The lacZ gene in the pGEM-T Easy vector allows blue-white screening to identify successful cloning of the RV2873 and Rv3875 fusion target genes. Transcription of the lacZ gene is initiated by IPTG [27]. LacZ encodes the enzyme β-galactosidase, breaking lactose into sugar components such as glucose and galactose. Blue bacterial colonies are formed when the enzyme β-galactosidase is present, which breaks down X-gal into galactose and 5-bromo-4-chloro-3-hydroxyindole, which produces a blue color. When blue colonies appear, the β-galactosidase enzyme works, meaning the lacZ gene is activated, thus indicating that DNA insertion into the vector has not occurred. In contrast, when Rv2873 and Rv3875 DNA were inserted into the multi-cloning site (MCS) vector, the lacZ gene was disrupted in the recombinant vector, preventing hydrolysis of the galactose substrate in the growth medium and causing the colonies to remain white. White bacterial colonies form when cells lack the enzyme β-galactosidase. Blue-white screening effectively identified successful transformants after cloning and transfecting the pGEM-T Easy-Rv2873 vector and the pGEM-T Easy-Rv2873 +Rv3875 recombinant plasmid into E. coli JM109 competent cells.

The construction of the recombinant vector, pGEM-T Easy- Rv2873+Rv3875, was achieved through the insertion of the DNA of the Rv3875 gene into the pGEM-T Easy- Rv2873 vector. The recombinant vector pGEM-T Easy-Rv2873+Rv3875 exhibited DNA fragments of 288 bp (from Rv3875 gen alone) and 3678 bp (from the pGEM-T Easy- Rv2873) upon double restriction with BamHI and HindIII enzymes. PCR colony technique and sequencing analysis also confirmed Rv2873 and Rv3875 genes insertion into pGEM-T Easy on the correct site and position in the cloning vector.

In this study, recombinant proteins MPT83 and ESAT6 were successfully produced in the E. coli BL-21 strain, forming white colonies. White colonies containing this recombinant fusion protein product were observed after E. coli B-L21 cells were transformed with the pTrcHisA-Rv2873+Rv3875 and cultured. The growth and synthesis of the recombinant protein were demonstrated by the visual change in the Luria-Bertani liquid medium from transparent to cloudy, which confirmed the success of the recombinant protein synthesis [19].

Cell walls, including cell membranes, can be efficiently disrupted by sonication. Biomolecules such as proteins, lipids (fats), and monosaccharides are released from the E. coli BL-21 strain cells during sonication and enter the clear liquid outside the cells. The presence of these biomolecules in the liquid would raise the fluid viscosity. Overall, the sonication process was employed to disrupt the bacterial cell walls and cell membranes, leading to the release of biomolecules from the E. coli BL-21 strain cells into the clear liquid [28]. This step is crucial for obtaining the desired proteins, MPT83 and ESAT6.

The use of IPTG as an inducer significantly enhances recombinant protein expression. In previous studies, the induction of recombinant proteins was found to be highly dependent on cell growth when inducers like IPTG were used. Higher IPTG concentrations could lead to increased recombinant protein production, but excessively high inducer concentrations could harm cell viability [30]. Another study reported that lesser doses of IPTG were preferable at high temperatures. The inducible expression of recombinant proteins and other target proteins in E. coli cells has led to IPTG's widespread use as an inducer in the scientific community [31]. Our result demonstrated that MPT83 and ESAT6 are both soluble proteins found inside cells. The protein was found to be quite prevalent in the supernatants but at lower concentrations in the pellet cells. This result showed that the E. coli successfully created a 28-kilo Dalton recombinant protein composing 6XHis-tagged MPT83 and ESAT6.

MPT83 has been demonstrated to elicit a significant and specific immunological response against M. tuberculosis. MPT83 could stimulate immune cells, including macrophages and dendritic cells, which in turn release cytokines like TNF-α and IFN-γ to initiate the adaptive immune response. So far, research on MPT83 as a seed vaccine candidate has been tested in the form of an RNA or DNA vaccine in mouse models and showed a specific MPT83 CD8+ T cell response [32]. Nonetheless, nucleic acid-based vaccines for TB have yet to be substantially examined, yet they may offer a framework for further vaccine design [33]. Besides, MPT83 and ESAT6 incorporated in Vaccine subunit H107, along with six other antigens not found in the BGC strain, have also been developed and tested in mouse models [34]. This vaccine's administration, along with BCG, enhances the adaptive response to M. tuberculosis infection, specifically in inducing a subset of under-differentiated CD4 Th1 cells that accumulate at the site of infection and persist post-Mtb challenge. Th1 with less differentiation may have a greater potential to develop memory T cells, which are essential for establishing sustained protection.

The achievement of creating this recombinant fusion protein offers new avenues for investigation. The limitation of this study is that it is only at the stage of fusion protein expression, and no research has yet been conducted to study the antibody response to the produced fusion antigen, either in vitro or in vivo. Future studies may use antigens obtained from this recombinant fusion protein to create TB vaccines, including the antibody response. This could be a promising direction for advancing TB vaccine research and development.

The PCR successfully amplified DNA bands from the genomic DNA of M. tuberculosis' Rv2873 and Rv3875 genes, which codes for the MPT83 plus ESAT6 fusion protein. These DNA fragments measured 948 bps in length and comprised the entire coding sequence for the MPT83 plus ESAT6 fusion protein down to its stop codon, TGA. Insertion of the Rv3875 gene, which codes for the ESAT6 protein, into the pGEM-T Easy Mpt83 plasmid yielded the pGEM-T Easy- Rv2873 +Rv3875 vector. As a result, the ESAT6 protein became fused with the MPT83 protein. Successful subcloning of the pGEM-T Easy- Rv2873 +Rv3875 plasmid into the pTrcHisA vector was achieved by employing nucleotide and amino acid sequence predictions of the 6XHis-tagged MPT83 plus ESAT6 recombinant proteins. E. coli BL-21   strain cells produced a 28-kilo Dalton fusion protein. The 28-kilo Dalton fusion protein was successfully produced in E. coli cells following amplification of the Rv2873 and Rv3875 genes from M. tuberculosis, construction of the pGEM-T Easy- Rv2873+Rv3875 vector, and subcloning of the fusion protein into the pTrcHisA vector. This study may advance our knowledge of MPT83 and ESAT6 fusion proteins, opening up their potential applications in future research, notably in vaccine development or other diagnostics and therapeutic approaches.

The authors would like to thank Mrs. Ermawati and Nadia from Hasanuddin University Biochemistry Laboratory for their technical help and TRG Biovax Tuberculosis for partially supporting this research. This study partially received funding from Fundamental Collaboration Research (PFK) from Hasanuddin University (Grant number 00309/UN4.22/PT.01.03/2024).

RBL, AA, AK, RA, RA, and NH collaborated to conduct practical research and laboratory techniques, gather and analyze data, and draft and edit the manuscript. MNM, AN, HK, and IH contributed to the critical reviewing and proofreading of this manuscript. All the authors have reviewed and approved the final version of the paper.

There is no conflict of interest among the authors.