Purification of recombinant human interleukin-3 expressed as inclusion bodies in <i>Escherichi coli</i>
Keywords:Escherichia coli JM109, amino acid sequence, inclusion body, protein refolding, protein purification, recombinant human IL-3.
Human interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. However, recombinant IL-3 is usually expressed as insoluble form (inclusion bodies) in Escherichia coli cells. This state of protein often shows no bioactivity. Herein, we report a simple method for solubilization, refolding and purification of recombinant human IL-3 expressed in E. coli cells. First, IL-3 was expressed in E. coli JM109 (DE3) after being induced with 0.05 mM IPTG at 25 oC. Under these conditions, IL-3 was produced as inclusion bodies with molecular weight of approximately 15 kDa on SDS-PAGE gel (14%). Next, IL-3 pellet was separated from the host soluble proteins using sonication followed centrifugation. Then, two strong denaturants such as urea or guanidine hydrochloride were used to test solubilization of the insoluble IL-3. After that, the resulting soluble IL-3 was renatured and subjected to gel filtration chromatography to collect purified IL-3 protein. Our results showed that fractionates contained a single band of IL-3 with recovery rate of about 30%. Several characteristics of recombinant IL-3 were then analyzed. The cytokine IL-3 showed its high purity with a sharp peak on RP-HPLC chromatagram. The Western blot showed a clear signal band on PVDF membrane to demonstrate its right antigenecity against human IL-3 antibody. Besides, amino acid sequence of this cytokine was confirmed by mass spectrophotometry method. The purified IL-3 cytokine is a potential material for further tests.
Clark S. C., Kamen R., 1987. The human hematopoietic colony-stimulating factors. Science 236: 1229–1237.
Dexter T. M., Spooncer E., 1987. Growth and differentiation in the hemopoietic system. Annu. Rev. Cell Biol., 3: 423–441.
Ding H., Griesel C., Nimtz M., Conradt H. S., Weich H.A., Jäger V., 2003. Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system. Protein Expr. Purif., 31: 34–41.
Duong Thu Huong, Nguyen Thi Quy, Dang Thi Ngoc Ha, Le Thi Thu Hong, Truong Nam Hai, 2016. Optimization of fermentation conditions for the expression of Interleukin-3 in fusion with PelB in E. coli. Tap chi Sinh hoc, 38(2): 250–256 (in Vietmamese with English summary).
Joseph B. C., Pichaimuthu S., Srimeenakshi S., Murthy M., Selvakumar K., Ganesan M., Manjunath S. R., 2015. An overview of the parameters for recombinant protein expression in Escherichia coli. J Cell Sci Ther., 6: 1–7.
Nguyen T. Q., Duong T. H., Dang T. N. H., Le N. G., Le Q. G., Do T. H., Nguyen V. D., Le T. T. H., Truong N. H., 2018. Enhanced soluble expression and efective purification of recombinant human interleukin-11 by SUMO fusion in Escherichia coli. Indian Journal of Biotechnology, 17: 579–585.
Sengupta P., Meena K., Mukherjee R., Jain S. K., Maithal K., 2008. Optimized conditions for high-level expression and purification of recombinant human interleukin-2 in E. coli. Indian J. Biochem. Biophys., 45: 91–97.
Singh S. M., Panda A. K., 2005. Solubilization and refolding of bacterial inclusion body proteins. J. Biosci. Bioeng., 99: 303–310.
Yang Y. C., Ciarletta A. B., Temple P. A., Chung M. P., Kovacic S., Witek-Giannotti J. S., Leary A. C., Kriz R., Donahue R. E., Wong G. G., 1986. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell, 47: 3–10.
Yun S. I., Yahya A. R. M., Cossar D., Anderson W. A., Scharer J. M., Moo-Young M., 2001. Temperature downshift increases recombinant cytokine titer in Streptomyces lividans fermentation. Biotechnol. Lett., 23: 1903-1905.
Ziltener H. J., Clark-Lewis I., Jones A. T., Dy M., 1994. Carbohydrate does not modulate the in vivo effects of injected interleukin-3. Exp. Hematol., 22: 1070–1075.