Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Abstract

Basic fibroblast growth factor (FGF-2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF-2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF-2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF-2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF-2 (42 mg/g dry cell) was achieved by fed-batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin-sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF-2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin-free and bioactive. It was successfully tested to keep primate embryonic stem cell and human-induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low-cost preparation of bioactive FGF-2 at bench-scale and may be beneficial to the effective production of other cytokines.