Pengaruh Suhu pada Ekspresi Pretrombin-2 Manusia Rekombinan di Escherichia coli ER2566

Saronom Silaban, Murniaty Simorangkir, Iman Permana Maksum, Khomaini Hasan

Abstract


Human recombinant prethrombin-2 (rhPT-2) was expressed in Escherichia coli. Previous studies reported that the expression of rhPT-2 in E. coli tends to formed inclusion body. This study aims to determine the effect of temperature on the expression of rhPT-2 in E. coli ER2566. This study begins with the isolation of recombinant plasmids, competent cell preparation and transformation of competent cells of E. coli ER2566 strain, and rhPT-2 gene expression with various induction temperatures 12, 18, 22 and 30°C. Characterization results showed that the induction temperature of 22°C was the optimum temperature of rhPT-2 gene expression as a soluble fraction in E. coli ER2566 with IPTG concentration 0.1 mM. These results prove that the temperature affects the expression of rhPT-2 in E. coli ER2566. [EFFECT OF TEMPERATURE ON RECOMBINANT HUMAN PRETROMBIN-2 EXPRESSION IN ESCHERICHIA COLI ER2566] (J. Sains Indon., 42(2): 25-30, 2018)

Keywords:
Induction Temperature, Soluble Fraction, Prethrombin-2, E. coli ER2566


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References


Baneyx, F., & Mujacic, M. (2004). Recombinant protein folding and misfolding in Escherichia coli. Nature Biotech, 22(11), 1399-408.

Cabrita, L. D., Dai, W., & Bottomley, S. P. (2006). A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotech, 6(1), 12.

Daly, R., & Hearn, M. T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol Recog, 18(2), 119-138.

Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotech Adv, 27(3), 297-306.

DiBella, E. E., Maurer, M. C., & Scheraga, H. A. (1995). Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J. Biol Chem, 270(1), 163-169.

Enus, S., Natadisastra, G., Shahib, M. N., & Sulaeman, R. (2011). Peran lem fibrin otologus pada penempelan tandur konjungtiva bulbi mata kelinci terhadap ekspresi gen fibronektin dan integrin. MKB, 43(4), 183-188.

Freydell, E. J., Ottens, M., Eppink, M., van Dedem, G., & van der Wielen, L. (2007). Efficient solubilization of inclusion bodies. Biotechnol. J, 2(6), 678-684.

Hartinger, D., Heinl, S., Schwartz, H. E., Grabherr, R., Schatzmayr, G., Haltrich, D., & Moll, W. D. (2010). Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B 1. Microb Cell Fac, 9(1), 62.

Heldebrant, C. M., Butkowski, R. J., Bajaj, S. P., & Mann, K. G. (1973). The Activation of Prothrombin II. Partial reactions, physical and chemical characterization of the intermediates of activation. J. Biol Chem, 248(20), 7149-7163.

Jonebring, A., Lange, U., Bucha, E., Deinum, J., Elg, M., & Lövgren, A. (2012). Expression and characterization of recombinant ecarin. Protein J, 31(5), 353-358.

Mann, K. G. (2011). Thrombin generation in hemorrhage control and vascular occlusion. Circulation, 124(2), 225-235.

Owen, W. G., Esmon, C. T., & Jackson, C. M. (1974). The conversion of prothrombin to thrombin I. Characterization of the reaction products formed during the activation of bovine prothrombin. J. Biol Chem, 249(2), 594-605.

Rizkia, P. R., Silaban, S., Hasan, K., Kamara, D. S., Subroto, T., Soemitro, S., & Maksum, I. P. (2015). Effect of Isopropyl-ß-D-thiogalactopyranoside concentration on prethrombin-2 recombinan gene expression in Escherichia coli ER2566. Procedia Chem, 17, 118-124.

Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. 3rd Cold Spring Harbor Laboratory Press. New York.

Silaban, S., Maksum, I. P., Ghaffar, S., Hasan, K., Enus, S., Subroto, T., & Soemitro, S. (2015). Codon optimization and chaperone assisted solubilization of recombinant human prethrombin-2 expressed in Escherichia coli. Microbiol Indones, 8(4), 177-182.

Silaban, S., Maksum, I. P., Enus, S., Hasan, K., Subroto, T., & Soemitro, S. (2016). Kajian ekspresi gen pretrombin-2 manusia sintetik pada Escherichia coli secara in silico untuk produksi trombin sebagai komponen lem fibrin. J. Pendidikan Kimia, 8(1), 58-64.

Silaban, S., Maksum, I. P., Hasan, K., Enus, S., Subroto, T., & Soemitro, S. (2017). Purification of recombinant human pretrombin-2 in Escherichia coli for thrombin production as fibrin glue components. J. Pendidikan Kimia, 9(1), 265-272.

Soejima, K., Mimura, N., Yonemura, H., Nakatake, H., Imamura, T., & Nozaki, C. (2001). An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived a-thrombin. J. Biochem, 130(2), 269-277.

So, I. S., Lee, S., Kim, S. W., Hahm, K. S., & Kim, J. (1992). Purification and activation of recombinant human prethrombin 2 produced in E. coli. Korean Biochem J, 25(1), 60-65.

Sørensen, H. P., & Mortensen, K. K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotech, 115(2), 113-128.

Subroto, T., Pertiwi, W., Fadhillah, M., Hasan, K., Budiantoro, O., Enus, S., & Soemitro, S. (2016). Cloning, expression, and functional characterization of autoactivated human prethrombin-2 synthetic gene by using Pichia pastoris SMD1168 as a host. Microbiol Indones, 10(2), 39-47.

Tegel, H. (2013). Proteome wide protein production (Doctoral dissertation, KTH Royal Institute of Technology).

Tripathi, N. K. (2009). High yield production of heterologous proteins with Escherichia coli. Def Sci J, 59(2), 137-146.

Yonemura H, Imamura T, Soejima K, Nakahara Y, Morikawa W, Ushio Y, Kamachi Y, Nakatake H, Sugawara K, Nakagaki T, Nozaki C. (2004). Preparation of recombinant a-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. J. Biochem, 135(5), 577-582.




DOI: https://doi.org/10.24114/jsi.v42i2.12245

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