Riyona Desvy Pratiwi, Asrul Muhamad Fuad


A synthetic human gene of  CSF3 (CSF3syn.Ec3), coding for hG-CSF was succesfully subcloned into pET32a(+) expression vector and fused with thioredoxin (Trx) at its N-terminal as fusion partner. The obtained fusion gene of Trx-CSF3syn within the recombinant plasmid pET32a(+)_CSF3yn.Ec3 was verified by PCR, plasmid restriction, and DNA sequencing analysis. In order to investigate the fusion gene expression, we transformed Escherichia coli BL21(DE3) as the host with the recombinant plasmid. The gene was succesfully expressed within the cytosol as fusion protein of Trx·tag, His·tag, S·tag, EK-site, and hG-CSF moities. By the autoinduction method, it was obtained 49% from the soluble fraction and 51% from the insoluble fraction. The soluble fraction was subsequently purified by IMAC method (Ni-NTA) and characterized.

Key words : hG-CSF, thioredoxin, autoinduction, IMAC, E.coli.

Full Text:



Awade, A.C., 1996, On hen egg fractionation : applications of liquid chromatography to the isolation and the purification of hen egg white and egg yolk proteins, Z Lebensm unters forsch, 202 : 1-14

Bartlow, P., Uechi, G.T., Cardamone Jr, J.J., Sultana, T., Fruchtl, M., Beitle, R.R., Ataai, M.M., 2011, Identification of native Escherichia coli BL21(DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contaminations pools with respect to recombinant protein expression level, Prot Expres Purif, 78 : 216-224

Blommel, P.G., Becker, K.J., Duvnjak, P., Fox, B.G, 2007, Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition, Biotechnol. Prog., 23, 585 – 598

Dale, D.C., 1998, The Discovery, Development and Clinical Applications of Granulocyte Colony Stimulating Factor, Trans Am Clin Climatol Assoc, 109 : 27-38

Das, K.M.P., Banerjee, S., Shekkar, N., Damodaran, K., Nair, R., Somani, S., Raiker, V., Jain, S., Padmanabhan, S., 2011, Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli, Int J Mol Sci, 12, 2064-2076

Dasari, V.K.R., Are, D., Joginapally, V.R., Mangamoori, L.N., Adibhatla, K.S.B.R., 2008, Optimization of the downstream process for high recovery of rhG-CSF, Process Biochem, 43 : 566-575

Deghani, S.A., Babeipour, V., Mofid, M.R., Faraji, F., 2010, An Efficient Prification Method for High Recovery of Recombinant Human Granulocyte Colony Stimulating Factor from Recombinant E.coli, Intl J. Env Sci Dev, 1 (2) : 111-114

Hill, C.P., T.D. Osslund, D. Eisenberg, 1993, The structure of granulocyte

colony-stimulating factor and its relationship to other growth factor, Prod Natl Acad Sci USA, 90: 5167--5171

Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March C.J., Cerretti, D.P., urdal, D.L., Conloon, P.J., 1988, A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purificaation, Nat Biotechnol, 6 : 1204 – 1210

Kang, Y.S., Song, J.A., Han, K.Y., Lee, J., 2014, Escherichia coli EDA is a novel fusion expression partner to improve solubility of aggregation-prone heterologous proteins, J Biotechnol,

LaValielli, E.R., Diblasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F., McCoy, M., 1993, A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E.coli Cytoplasm, Nat Biotechnol, 10 : 187-193

Li, Z., Kessler, W., van den Heuvel, J., Rinas, U., 2011, Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. Appl Microbiol Biotechnol, 91 : 1203-1213

Metcalf, D., 1987, The Role of The Colony-Stimulating Factors in Resistance to Acute Infections, Immunol Cell Biol, 65 : 35-45

Nagata, 1986, The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor, The EMBO Journal, 5(3) : 575-581

Porath, J., 1992, A review – immobilized metal ion affinity chromatography, Prot Expres Purif, 3 : 263-281

Pratiwi, R.D., Fuad, A.M., 2012, Influence of temperature on recombinant granulocyte colony stimulating factor production by Escherichia coli BL21(DE3) expression system using autoinduction, in Proceedings- International Conference on Biotechnology 2012, ed. Prasetyoputri, A., et al, Research Center for Biotechnology, Indonesian Institute of Sciences, Bogor, Indonesia, pp. 419-427

Sambrook, J.,and Russell, D.W., 2001, The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells,. In Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor, New York. ,p. 116-118

Sandhev, D., Chrigwin, J.M., 1998, Solubility of Protein Isolated from Inclusion Bodies Is Enhanced by Fusion to Maltose-Binding Protein or Thioredoxin, Prot Express Purif, 12 : 122-132

Song, J.A., Lee, D.S., Park, J.S., Han, K.Y., Lee, J., 2011, A novel Escherichia coli solubility enhancer protein for fusion expression of aggregation-prone heterologous proteins, Enzyme Microbiol Technol, 49, 124-130

Souza, L.M.,Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., Chen, K.K., Barendt, J., Platzer, E., Moore, M.A.S., Meterlsmann, R., Welte, K.,1986, Recombinant human granulocyte colony-stimulating factor : effect on normal and leukemic myeloid cells, Science, Vol. 232 no.4746 : 61-65

Studier, 2005, Protein production by auto-induction in high density shaking cultures, Protein Expres Purif, 41 : 207-234

Thatcher, D.R., 1990, Recovery of Theurapetic Proteins from Inclusion Bodies : Problems and Process Strategies, Biochem Soc Trans, 18(2) : 234-235

Tyler, R.C., Sreenath, H.K., Singh, S., Aceti, D.J., Bingman, C.A., Markley, J.L., Fox, B.G., 2005, Auto-induction medium for the production of [U-15N]-and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination, Protein Expr Purif, 40, 268-278

Welte, K., 2012, Discovery of G-CSF and early clinical studies, in Twenty Years of G-CSF, Milestone in Drug Therapy, edited by G. Molineux, et al, DOI 10.1007/978-3-0348-0218-5_2

Wingfield, P., Benedict, R., Turcatti, G., Allet, B., Mermod, J.J., Delamarter, J., Simona, M.G., Rose, K., 1988, Characterization of Recombinant Derived Granulocyte-colony stimulating factor (G-CSF), Biochem. J., 256 : 213-218

Wulandari, S., 2010, Subkloning Gen Sintetik CSF3syn (Colony Stimulating Factor-3) pada Vektor Ekspresi pET32a(+) dan Transformasi Vektor Rekombinan pada Escherichia coli BL21(DE3)pLysS, Skripsi, Universitas Indonesia, Indonesia

Yamamoto, A., Iwata, A., Saitoh, T., Tuchiya, K., Kanai, T., Tsujimoto, H., Hasegawa, A., Ishihama, A., Ueda, S., 2002, Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor, Vet Immunol Immunop, 90 : 169-177

Yasukawa, T., Ishii, C.K., Maekawa, T., Fujimoto, J., Yamamoto, T., Ishii, S., 1995, Increase of Solubility of Foreign Proteins in Escherichia coli by Coproduction of the Bacterial Thioredoxin, J Biol Chem, 43 : 25328-25331



  • There are currently no refbacks.


Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Indonesian J Pharm indexed by:


analytics View My Stats