The Effect of Single Co-expression of The DnaK-DnaJ-GrpE and GroEL/ES Chaperones and Their Combination on Expression Intein-pretrombin-2 in Escherichia coli ER2566

Iman Permana Maksum, D Agus Yusuf Wildan, Khomaini Hasan, Toto Subroto

Abstract


The use of recombinant thrombin in the manufacture of fibrin glue allows diseases contamination to be avoided. However, the expression of recombinant protein in E. coli still has a disadvantage of the formation of inclusion bodies, so it needs to be minimized by co-expression of chaperones. Therefore, the aim of this study was to determine the effect of single DnaK-DnaJ-GrpE and GroEL/ES chaperone expression and their combination on the expression of intein-pretrombin-2Ti,pH on E. coli ER2566. The method started with isolation of pTWIN1-prethrombin-2Ti,pH and pG-KJE8 from E. coli TOP10F' and DH5α respectively, the co-transformation of the expression host E. coli ER2566 using pG-KJE8 and pTWIN1-prethrombin-2Ti,pHvectors, the chaperone co-expression was induced using L-Arabinosa before IPTG induction and cell culture growth was incubated at 22 oC. The expression products were characterized by using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results of the co-expression of chaperone showed that the number of soluble fraction was higher than the one without co-expression of chaperone. In addition, the co-expression of chaperone using pG-KJE8 in intein-prothrombin-2Ti,pH expression was sufficient using tetracycline as an inducer.


Keywords


Intein-pretrombin-2Ti; pH; chaperone co-expression of DnaK-DnaJ-GrpE and GroEL/ES; E. coli ER2566

References


Alibolandi M, Mirzahoseini H, Nehi FM, Tabatabaian G. 2010. Improving recombinant protein solubility in Escherichia coli : Identification of best chaperone combination which assists folding of human basic fibroblast growth factor. African Journal of Biotechnology. 9(47): 8100–8109.

Baneyx F. 1999. Recombinant protein expression in Escherichia coli. Current opinion in biotechnology. 10(5): 411–421.

Baneyx F, Mujacic M. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology. 22(11): 1399–1407.

Choi JH, Lee SY. 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology. 64(5): 625–635.

Enus S, Dalimoenthe NZ, Kartiwa A. 2006. Teknik lem fibrin otologus pada cangkok konjungtiva bulbi mata kelinci bulbar conjunctival graft using an autologous fibrin glue in rabbit eyes. Majalah Kedokteran Bandung. 41(4): 169–173.

Garcia-carbonero R, Carnero A, Paz-ares L. 2013. Inhibition of HSP90 molecular chaperones : moving into. Lancet Oncology. 14(9): e358–e369.

Gupta SRVKA. 2002. Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli. Applied Microbiology and Biotechnology. 58(3): 330–337.

Henderson B, Pockley AG. 2010. Molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation. Journal of Leukocyte Biology. 88(3): 445–462.

Indriyani A, Anggraeni NI, Sriwidodo, Maksum IP. 2019. Optimization extracellular secretion of recombinant human epidermal growth factor (hEGF) in Escherichia coli BL21 (DE3) pd881-ompa-hegf by using response surface method (RSM). International Journal of Research in Pharmaceutical Sciences. 10(3): 1824–1831.

Indriyani A, Gaffar S, Latifah FPU, Maksum IP. 2020. Co-expression of recombinant human epidermal growth factor (rhEGF) In Escherichia coli BL21 (DE3) with Bacillus cereus phospholipase C. In AIP Proceeding.

Juozas Š, Neubauer P. 2011. Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression. Microbial Cell Factories. 10(1): 1–11.

Latifah FPU, Indriyani A, Pratiwi R, Sriwidodo, Maksum IP. 2020. Effects of growth medium on extracellular secretion of human epidermal growth factor in Escherichia coli by co-expression with Bacillus cereus Phospholipase C. In IOP Conference Series.

Maksum IP, Sriwidodo S, Yosua Y. 2019. Strategi Peningkatan Ekspresi Protein Rekombinan Secara Intraselular pada Inang. Sumedang: Alqaprint.

Maksum IP, Utama E, Sriwidodo S, Subroto T. 2017. Extracellular secretion of recombinant human epidermal growth factor by using trimethylamine N-Oxide reductase a (TORA) signal peptide in escherichia coli BL21 (DE3). Journal of Pharmaceutical Sciences and Research. 9(6): 1007–1016.

Maksum IP, Sriwidodo, Gaffar S, Hasan K, Subroto T, Soemitro S. 2017. Teknik Biologi Molekular. Sumedang: Alqaprint.

Maksum IP, Sriwidodo, Indriyani A. 2017. Sistem Ekspresi Protein Rekombinan dalam Escherichia coli secara Ekstraselular. Sumedang: Alqaprint.

Melati R, Indriyani A, Gaffar S, Sriwidodo S, Maksum IP. 2019. Comparison of extracelullar secretion of recombinant human epidermal growth factor using TorA and PelB signal peptides in Escherichia coli BL21 (DE3). Asian Journal of Pharmaceutical and Clinical Research. 12(11): 81–84.

Rizkia PR, Silaban S, Hasan K, Kamara DS, Subroto T, Soemitro S, Maksum IP. 2015. Effect of isopropyl-β-D-thiogalactopyranoside concentration on prethrombin-2 recombinan gene expression in Escherichia coli ER2566. Procedia Chemistry. 17(2015): 118–124.

Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. 2019a. Construction and optimization of prethrombin-2 human genes in e. coli for the production of active thrombin. Journal of Physics: Conference Series. 1374: 12047.

Silaban S, Gaffar S, Simorangkir M, Maksum IP, Subroto T. 2019b. Effect of IPTG concentration on recombinant human prethrombin-2 expression in escherichia coli BL21(DE3) arctic express. IOP Conference Series: Earth and Environmental Science. 217: 12039.

Silaban S, Maksum IP, Enus S. 2016. Kajian ekspresi gen pretrombin-2 manusia sintetik pada Escherichia coli secara in silico untuk produksi trombin. Jurnal Pendidikan Kimia. 8(1): 58–64.

Silaban S, Maksum IP, Ghaffar S, Hasan K, Enus S, Subroto T. 2014. Codon optimization and chaperone assisted solubilization of recombinant human prethrombin-2 expressed in Escherichia coli. Microbiology Indonesia. 8(4): 170–175.

Silaban, Saronom, Simorangkir M, Gaffar S, Maksum I, Subroto T. 2019. Temperature effect on expression of recombinant human prethrombin-2 in Escherichia coli BL21(DE3) Arctic Express. Jurnal Pendidikan Kimia. 11(3): 122–128.

Siurkus J. 2013. Approach for production of sensitive to oxidation and aggregating proteins in E. coli at the example of a heterologous ribonuclease inhibitor.

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 alpha thrombin. Journal of Biochemistry. 130(2): 269–277.

Sriwidodo S, Maksum IP, Riswanto N, Rostinawati T, Subroto T. 2017. Extracellular secretion recombinant of human epidermal growth factor (hEGF) using pectate lyase B (PelB) signal peptide in Escherichia coli BL21(DE3). International Journal of Research in Pharmaceutical Sciences. 8(1): 33–40.

Yan X, Hu S, Guan YX, Yao SJ. 2012. Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli. Applied Microbiology and Biotechnology. 93(3): 1065–1074.


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DOI: 10.15408/jkv.v6i1.11333

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