Expression of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B in Escherichia coli and Its Recovery from Inclusion Body

Lalu Rudyat Telly Savalas, Prapti Sedijani, Saprizal Hadisaputra, Jannatin Ardhuha, Chomsa Asih Lestari, Etty Nurul Wahidah

Abstract


The present study aims at expressing and partially purifying PtpB in active form. To achieve this, Mtb PtpB gene has been cloned into pET30a vector and overexpressed in Escherichia coli BL 21(DE3) under IPTG induction in the form of an inclusion body. Following resolubilization by urea and dialysis, the resulted PtpB has been shown to be active against para-Nitrophenyl phosphate. It is concluded that the resulted PtpB has had been recovered from inclusion body to give the active form of the enzyme, and thus the success in overexpressing PtpB provides the required material to investigate the biochemical properties of the pathogen virulence factor further.


Keywords


Latent infection; Mycobacterium tuberculosis; PtpB; Overexpression

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References


Ahmad, S. (2011). Pathogenesis, immunology, and diagnosis of latent Mycobacterium tuberculosis infection. Clinical and Developmental Immunology, 2011.

Bash, E. (2015). Latent tuberculosis infection. Ph.D. Proposal, 1(5), 6266.

Boshoff, H. I. M., & Barry, C. E. (2005). Tuberculosis metabolism and respiration in the absence of growth. Nature Reviews Microbiology, 3(1), 7080.

Chapman, H. J., & Lauzardo, M. (2014). Advances in diagnosis and treatment of latent tuberculosis infection. Journal of the American Board of Family Medicine, 27(5), 70412.

Chauhan, P., Reddy, P. V., Singh, R., Jaisinghani, N., Gandotra, S., & Tyagi, A. K. (2013). Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs. PLoS ONE, 8(10),

Chen, D., Chen, H., She, Z., & Lu, Y. (2016). Identification of Bostrycin Derivatives as Potential Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatase (MptpB). Medicinal Chemistry, 12(3), 296302.

Chiaradia, L. D., Mascarello, A., Purificao, M., Vernal, J., Cordeiro, M. N. S., Zenteno, M. E., Terenzi, H. (2008). Synthetic chalcones as efficient inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase PtpA. Bioorganic and Medicinal Chemistry Letters, 18(23), 62276230.

Collins, D., Hafidz, F., & Mustikawati, D. (2017). The economic burden of tuberculosis in Indonesia. The International Journal of Tuberculosis and Lung Disease, 21(9), 2017.

Dhanjal, J. K., Grover, S., Sharma, S., Singh, A., & Grover, A. (2014). Structural insights into mode of actions of novel natural Mycobacterium protein tyrosine phosphatase B inhibitors. BMC Genomics, 15 Suppl 1(Suppl 1), S3.

Forrellad, M. A., Klepp, L. I., Gioffr, A., Sabio y Garca, J., Morbidoni, H. R., de la Paz Santangelo, M., Bigi, F. (2013). Virulence factors of the Mycobacterium tuberculosis complex. Virulence, 4(1), 366.

Grundner, C., Ng, H. L., & Alber, T. (2005). Mycobacterium tuberculosis protein tyrosine phosphatase PtpB structure reveals a diverged fold and a buried active site. Structure, 13(11), 16251634.

He, R., Yu, Z. H., Zhang, R. Y., Wu, L., Gunawan, A. M., & Zhang, Z. Y. (2015). Cefsulodin Inspired Potent and Selective Inhibitors of mPTPB, a Virulent Phosphatase from Mycobacterium tuberculosis. ACS Medicinal Chemistry Letters, 6(12), 12311235.

Li, Y., Wang, B., Zhang, H., Wang, Z., Zhu, S., & Ma, H. (2015). High-level expression of angiotensin converting enzyme inhibitory peptide Tuna AI as tandem multimer in Escherichia coli BL21 (DE3). Process Biochemistry, 50(4), 545552.

Lillebaek, T., Dirksen, A., Baess, I., Strunge, B., & Thomsen, V. . (2002). Molecular Evidence of Endogenous Reactivation of Mycobacterium tuberculosis after 33 Years of Latent Infection. The Journal of Infectious Diseases, 185(3), 401-404.

Lorenz, U. (2011). Protein Tyrosine Phosphatase Assays. Curr Protoc Immunol., (434), 114.

Mascarello, A., Mori, M., Chiaradia-Delatorre, L. D., Menegatti, A. C. O., de Monache, F., Ferrari, F., Botta, M. (2013). Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products. PLoS ONE, 8(10), 112.

Palmer, I., & Wingfield, P. T. (2004). Preparation and extraction of insoluble (Inclusion body) proteins from Escherichia coli. Current Protocols in Protein Science, (8 M), 125.

Palomino, J. C., & Martin, A. (2016). The potential role of trimethop sulfamethoxazole in the treatment of drug-resistant tuberculosis. Future microbiology, 11(4), 539-547.

Parida, S. K., & Kaufmann, S. H. E. (2010). Novel tuberculosis vaccines on the horizon. Current Opinion in Immunology, 22(3), 374384.

Studier, F. W., & Moffat, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of molecular biology, 189(1), 113-130.

Sundaramurthy, V., & Pieters, J. (2007). Interactions of pathogenic mycobacteria with host macrophages. Microbes and Infection, 9(1415), 16711679.

Wahyuningsih, S. P. A., Pramudya, M., & Sugiharto, S. (2016). Influence of Polysaccharide Krestin from Coriolus versicolor Extract on Nitrite and Malondialdehyde Concencentrations of Mus musculus Serum Exposed by Mycobacterium tuberculosis. Biosaintifika: Journal of Biology & Biology Education, 8(1), 12-17.

Zhou, B., He, Y., Zhang, X., Xu, J., Luo, Y., Wang, Y., Zhang, Z.-Y. (2010). Targeting mycobacterium protein tyrosine phosphatase B for antituberculosis agents. Proceedings of the National Academy of Sciences of the United States of America, 107(10), 45738.




DOI: https://doi.org/10.15294/biosaintifika.v9i3.12384

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