Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

Dian Fitria Agustiyanti, Debbie Sofie Retnoningrum, Heni Rachmawati, Asrul Muhamad Fuad


Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 


G-CSF • Thioredoxin • Soluble protein • E. coli

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Alrokayan, S.. (2011). Chemical Synthesis and Improved Expression of Recombinant human Granulocyte Colony Stimulating Factor cDna. Genetics and Molecular Research. 10 (4): 2671-2678, http://dx.doi.org/10.4238/2011.October.31.18.

Babaeipour, V., Abbas, M.P.H., Sahebnazar, Z., and Alizadeh, R.. (2010). Enhancement of human Granulocyte-Colony Stimulating Factor Production in Recombinant E. coli using Batch Cultivation. Bioprocess and Biosystems Engineering. 33: 591-598


Bayer, M.E.. (1968). Areas of Adhesion Between Wall and Membrane of Escherichia coli

Journal of General Microbiology. 53: 395-404


Bonig, H.B. Hannen, M., lex, C., Wolfel, S., Banning, U., and Nurnberger, W.. (1999). Additive Effects of Infection and Neutropenia on The Induction of Granulocytopoietic Activity in vivo. Cancer. 86 (2): 340-348.

Bringans, S., Eriksen, S., Kendrick, T., Gopalakrishnakone, P., Livk, A., Lock, R., and Lipscombe, R.. (2008). Proteomic Analysis of the Venom of Heteronometrus longimanus (Asian Black Scorpion). Proteomics. 8: 1081-1095


Callard, R. E. and A. J. H. Gearing. (1994). The Cytokine Facts book. Academic Press, Inc., San Diego, California.

Chiti, F., Stefani, M., Taddei, N., Ramponi, G., and Dobson, C.M... (2003). Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature. 424: 805 -808.

Dehaghani, S.A., Babaeipour, V., mofid, M.R., Divsalar, A., and Faraji, F.. (2010). An Efficient Purification Method for High Recovery of Recombinant human Granulocyte Colony Stimulating Factor from Recombinant E. coli. International Journal of Environmental Science and Development. 1(2): 111-114.

Devlin, P.E., Drummond, R.J., Toy, P., Mark, D.F., Watt, K.W., and Devlin, J.J.. (1998). Alteration of Amino-Terminal Codons of human Granulocyte Colony-Stimulating Factor Increases Expression Levels and Allows Efficient Processing by Methionine Aminopeptidase in Escherichia coli. Gene. 65: 13-22.

Fallah, M.J., Akbari, B., Saeedinia, A.R., Karimi, M., Vaez, M., Zeinoddini, M., Soleimani, and M., Maghsoudi, N.. (2003). Overexpression of Recombinant human Granulocyte Colony-Stimulating Factor in E. coli. International Journal of Molecular Sciences. 28 (3): 131-134.

Fuad, A. M., Yuliawati, and Agustiyanti, D.F.. (2013). Desain dan konstruksi gen CSF3 Sintetik (CSF3syn) mengandung kodon preferensi Escherichia coli dengan teknik PCR. Proceeding of national seminar XXII ”Kimia dalam Industri dan Lingkungan”. 87-98.

ISSN : 0854-4778

Hill, C.P., Osslund, T.D., and Eisenberg, D.. (1993). The Structure of Granulocyte Colony Stimulating Factor and It’s Relationship to Other Growth Factor. Proceeding of National Academy of Science, USA. 90: 5167-5171.

Jeong, K.J. and Lee, S.Y.. (2001) Secretory Production of human Granulocyte Colony-Stimulating Factor in Escherichia coli

Protein expression and purification. 23: 311-318.


Johnson, B.H. and Hecht, M.H.. (1994). Recombinant Protein can be Isolated from E. coli by Repeated Cycles of Freezing and Thawing. Biotechnology. 12: 1357-1360,


Khow, O. and Suntrarachun, S.. (2012). Strategies for Production of Active Eukaryotic Proteins in Bacterial Expression System. Asian pasific journal of tropical biomedicine.159 – 162


Kumar, J.K., Tabor, S., and Richardson, C.C.. (2003). Proteomic Analysis of Thioredoxin-Targeted Proteins in Escherichia coli. The National Academy of Sciences of the USA.


Lilie, H., E. Schawrz, R. and Rudolph. (1998). Advances in Refolding of Proteins Produced in E. coli. Current Opinion in Biotechnology. 9: 497-501.

Lu, S., Boone, T., Souza, L., and Lai, P.. (1989). Disulfide and Secondary Structure of Recombinant human Granulocyte Colony-Stimulating Factor. Archives of Biochemistry and Biophysics. 1: 81-92.

Rao, D.V.K., Narasu, M.L., and Rao, A.K.S.B.. (2008). A Purification Method for Improving the Process Yield and Quality of Recombinant human Granulocyte Colony-Stimulating Factor Expressed in Escherichia coli and It’s Characterization. Biotechnology and Applied Biochemistry.50: 77-87.


Ritz, D. and beckwith, J.. (2001). Roles of Thiol-Redox Pathways in Bacteria. Annual Review of Microbiology. 55: 21-48.


Rodriguez, J., Gupta, Nisin., Smith, Richard D., Pevzner, Pavel A.. (2008). Does Trypsin cut Before Proline?. Journal of Proteome Research. 7 (1) : 300 – 305.


Song, J.A., Han, K.Y., Ahn, K.Y., Park, J.S., Seo, H.S., and Lee, J.. (2009). Proteolysis and Synthetic Strategy of Human G-CSF in Escherichia coli BL21(DE3). Enzyme and Microbial Technology. 45: 7-14


Vanz, A. LS., Gaby R., Mario S.P., Jocelei M.C., Sergio L. D., Luiz A. B., and Diego S. S.. (2008). Human Granulocyte Colony Stimulating Factor (hG-CSF) : Cloning, Overexpression, Purification & Characterization. Microbial Cell Factories. 7 - 13


Welte, K., Gabriel, J., Bronchud, M.H., Platzer, E., and Morstyn, G.. (1996). Filgrastim (r-met-HuG-CSF) : The First 10 years. Blood. 88: 1907 – 1929.

Yasukawa, T., C. Kanei-Ishii, T. Maekawa, J. Fujimoto, and T. Yamamoto, S. Ishii. (1995). Increase of Solubility of Foreign Proteins in Escherichia coli by Coproduction of Bacterial Thioredoxin. The Journal of Biological Chemistry. 270: 25328-25331.

DOI: http://dx.doi.org/10.14203/ann.bogor.2017.v21.n1.1-8


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