Appl Biochem Biotechnol (2014) 172:2425–2435 DOI 10.1007/s12010-013-0708-y
Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli Chang-Kyu Kim & Jun-Ha Choi & Seung-Bae Lee & Sang-Mahn Lee & Jae-Wook Oh
Received: 24 October 2013 / Accepted: 25 December 2013 / Published online: 5 January 2014 # Springer Science+Business Media New York 2014
Abstract Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99 % and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals. Keywords Human granulocyte colony-stimulating factor . Fed-batch . Inclusion bodies . Refolding buffer . Recombinant protein purification
Introduction Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that is secreted in response to specific stimulation by a variety of cells including macrophages, fibroblasts, endothelial cells, and bone marrow stroma [1, 2]. Upon binding to the cognate membrane receptor on cells, G-
Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0708-y) contains supplementary material, which is available to authorized users.
C.
Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli Chang-Kyu Kim & Jun-Ha Choi & Seung-Bae Lee & Sang-Mahn Lee & Jae-Wook Oh
Received: 24 October 2013 / Accepted: 25 December 2013 / Published online: 5 January 2014 # Springer Science+Business Media New York 2014
Abstract Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99 % and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals. Keywords Human granulocyte colony-stimulating factor . Fed-batch . Inclusion bodies . Refolding buffer . Recombinant protein purification
Introduction Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein that is secreted in response to specific stimulation by a variety of cells including macrophages, fibroblasts, endothelial cells, and bone marrow stroma [1, 2]. Upon binding to the cognate membrane receptor on cells, G-
Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0708-y) contains supplementary material, which is available to authorized users.
C.
