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Temperature effect on expression of recombinant human prethrombin-2 in Escherichia coli BL21(DE3) ArcticExpress
Abstract
Many proteins produced in E. coli accumulate in inclusion bodies. This study aims to detect the role of temperature in reducing the formation of inclusion bodies during recombinant human prethrombin-2expressed in E. coli BL21 (DE3) Arctic Express host. In this study, we created a host growth curve to find out the right time to add an inducer. The inducer used in our experiment was IPTG 0.1 mM. The fermentation process use a temperature of 12°C and 22°C. The results showed that recombinant human prethrombin-2 was successfully expressed as protein soluble using an optimum temperature of 12°C in E. coli BL21 (DE3) Arctic Express. It was indicated from the 63kDa protein band obtained from the soluble fraction on SDS-PAGE. The higher temperature of fermentation increased the amount of protein in the insoluble fraction due. It concluded that the fermentation temperature affect the rate of prethrombin-2 expression.
Keywords: E. coli BL21(DE3) ArcticExpress, prethrombin-2, soluble, temperature
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Bishop, P., Lewis, K., Schultz, J., & Walker, K. (2006). Comparison of recombinant human thrombin and plasma-derived human α-thrombin. Seminars in Thrombosis and Hemostasis, 32(S 1), 086–097, doi: 10.1055/s-2006-939558
Bollag, D. M., Rozycki, M. D., & Edelstein, S. J. (1996). Protein method. 2nd, John Wiley & Sons, Inc. USA.
Bonatti, J. A., Bechara, S. J., Dall’Col, M. W. L., Cresta, F. B., Carricondo, P. C., & Kara-José, N. (2007). A fibrin-related line of research and theoretical possibilities for the use of fibrin glue as a temporary basal membrane in non-perforated corneal ulcers and in photorefractive keratectomy (PRK)-operated corneas. Arquivos Brasileiros de Oftalmologia, 70(5), 884–889, doi: 10.1590/s0004-27492007000500029
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 Biotechnology, 6, 12, doi: 10.1186/1472-6750-6-12
Choi, E. H. (1989). Cloning and expression of human prethrombin 2 cDNA in Escherichia coli. Korean Biochem. J., 22, 154-160.
Chen, R. (2012). Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnology Advances, 30(5), 1102–1107, doi: 10.1016/j.biotechadv.2011.09.013
Côté, H. C., Stevens, W. K., Bajzar, L., Banfield, D. K., Nesheim, M. E., & MacGillivray, R. T. (1994). Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and R284A). Journal of Biological Chemistry, 269(15), 11374-11380.
DiBella, E. E., Maurer, M. C., & Scheraga, H. A. (1995). Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. Journal of Biological Chemistry, 270(1), 163–169, doi: 10.1074/jbc.270.1.163
Ferrer, M., Chernikova, T. N., Yakimov, M. M., Golyshin, P. N., & Timmis, K. N. (2003). Chaperonins govern growth of Escherichia coli at low temperatures. Nature Biotechnology, 21(11), 1267–1267, doi: 10.1038/nbt1103-1266b
Ferrer, M., Chernikova, T. N., Timmis, K. N., & Golyshin, P. N. (2004). Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain. Applied and Environmental Microbiology, 70(8), 4499–4504, doi: 10.1128/aem.70.8.4499-4504.2004
Freydell, E. J., Ottens, M., Eppink, M., van Dedem, G., & van der Wielen, L. (2007). Efficient solubilization of inclusion bodies. Biotechnology Journal: Healthcare Nutrition Technology, 2(6), 678-684, doi: 10.1002/biot.200700046
Hartinger, D., Heinl, S., Schwartz, H., 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 B1. Microbial Cell Factories, 9(1), 62, doi: 10.1186/1475-2859-9-62
Jonebring, A., Lange, U., Bucha, E., Deinum, J., Elg, M., & Lövgren, A. (2012). Expression and characterization of recombinant ecarin. The Protein Journal, 31(5), 353–358, doi: 10.1007/s10930-012-9409-6
Laurens, N., Koolwijk, P. D., & De Maat, M. P. M. (2006). Fibrin structure and wound healing. Journal of Thrombosis and Haemostasis, 4(5), 932-939, doi: 10.1111/j.1538-7836.2006.01861.x
Moradian, C., Fazeli, M., & Abedi, D. (2013). Over expression of the Interferon β-1b by optimizing induction conditions using response surface meth-odology. Journal of Biology and Today’s World, 2(4), doi: 10.15412/j.jbtw.01020401
Osadská, M., Boňková, H., Krahulec, J., Stuchlík, S., & Turňa, J. (2014). Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli. Applied Microbiology and Biotechnology, 98(22), 9259–9270, doi: 10.1007/s00253-014-5840-2
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. Journal of Biological Chemistry, 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-β-D-thiogalactopyranoside concentration on prethrombin-2 recombinan gene Expression in Escherichia coli ER2566. Procedia Chemistry, 17, 118–124, doi: 10.1016/j.proche.2015.12.121
Russo, G., Gast, A., Schlaeger, E.-J., Angiolillo, A., & Pietropaolo, C. (1997). Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin. Protein Expression and Purification, 10(2), 214–225, doi: 10.1006/prep.1997.0733
Sambrook, J. E. F., & Russell, D. W. (2001). Molecular cloning: A laboratory manual. 3rd Ed. Cold Spring Harbor Laboratory Press. New York.
Silaban, S., Maksum, I. P., Ghaffar, S., Hasan, K., Enus, S., Subroto, T., & Soemitro, S. (2014). Codon optimization and chaperone assisted solubilization of recombinant human prethrombin-2 expressed in Escherichia coli. Microbiology Indonesia, 8(4), 177–182, doi: 10.5454/mi.8.4.5
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, doi: 10.24114/jpkim.v9i1.6201
Silaban, S., Gaffar, S., Simorangkir, M., Maksum, I. P., & Subroto, T. (2018). Effect of IPTG Concentration on Recombinant Human Prethrombin-2 Expression in Escherichia coli BL21(DE3) ArcticExpress. IOP Conference Series: Earth and Environmental Science, 217, 012039, doi: 10.1088/1755-1315/217/1/012039
Silaban, S., Gaffar, S., Simorangkir, M., Maksum, I. P., & Subroto, T. (2019). Construction and optimization of prethrombin-2 human genes in E. coli for the production of active thrombin. Journal of Physics: Conference Series, 1374, 012047, doi :10.1088/1742-6596/1374/1/012047
Singh, S. M., & Panda, A. K. (2005). Solubilization and refolding of bacterial inclusion body proteins. Journal of Bioscience and Bioengineering, 99(4), 303–310, doi: 10.1263/jbb.99.303
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 Biochemical Journal, 25(1), 60-65.
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 α-thrombin. Journal of Biochemistry, 130(2), 269–277, doi: 10.1093/oxfordjournals.jbchem.a002982
Spotnitz, W. D., & Prabhu, R. (2005). Fibrin sealant tissue adhesive - Review and update. Journal of Long-Term Effects of Medical Implants, 15(3), 245–270, doi: 10.1615/jlongtermeffmedimplants.v15.i3.20
Sørensen, H. P., & Mortensen, K. K. (2005). Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology, 115(2), 113–128, doi: 10.1016/j.jbiotec.2004.08.004
Strocchi, M., Ferrer, M., Timmis, K. N., & Golyshin, P. N. (2006). Low temperature-induced systems failure inEscherichia coli: Insights from rescue by cold-adapted chaperones. Proteomics, 6(1), 193–206, doi: 10.1002/pmic.200500031
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. Microbiology Indonesia, 10(2), 39–47, doi: 10.5454/mi.10.2.1
Wang, Y., Wang, Z., Duo, Y., Wang, X., Chen, J., & Chen, J. (2018). Gene cloning, expression, and reducing property enhancement of nitrous oxide reductase from Alcaligenes denitrificans strain TB. Environmental Pollution, 239, 43–52, doi: 10.1016/j.envpol.2018.04.005
Yonemura, H., Takayuki, I., Kenji, S., Yo, N., Wataru, M., Yoshitaka, U., Yasuharu, K., Hiroshi, N., Keishin, S., Tomohiro, N., & Chikateru, N. (2004). Preparation of recombinant α-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. J. Biochem, 135(5), 577-582, doi: 10.1093/jb/mvh070
DOI: https://doi.org/10.24114/jpkim.v11i3.15779
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