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.

Fibroblast growth factor 2 dimer with superagonist in vitro activity improves granulation tissue formation during wound healing

“…This mutation was made on a pET29c(+)hFGF-2 plasmid provided by the Helmholtz Centre for Infection Research, Braunschweig, Germany[60]. Employing E. coli host BL21(DE3), the mutant was expressed and purified as previously described.…”

Fast and Efficient Multitransgenic Modification of Human Pluripotent Stem Cells

“…Human ES cell lines hES3 (Reubinoff et al, 2000) and I3 (Amit and Itskovitz-Eldor, 2002), as well as the three human iPSC lines hCBiPS2 (cord blood endothelial cell derived; Haase et al, 2009), hFF1-iPS4 (human foreskin fibroblast derived), and hHSC_F1285_T-iPS2 (hematopoietic stem cell derived; Hartung et al, 2013), were cultured on irradiated murine embryonic fibroblasts (MEFs) in 80% KnockOut DMEM (Dulbecco's modified Eagle's medium) supplemented with 20% KnockOut Serum Replacement, 1 mM l-glutamine, 0.1 mM 2-mercaptoethanol, 1% nonessential amino acid stock (all from Life Technologies, Karlsruhe, Germany), and basic fibroblast growth factor (bFGF) at either 50 ng/ml (hES3, I3) or 4 ng/ml (hiPSCs) (supplied by the Institute for Technical Chemistry, Leibniz University Hannover, Hannover, Germany) (Chen et al, 2012). Cells were cultured in 6-well plates and passaged once per week with collagenase type IV (1 mg/ml; Invitrogen).…”

One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers

“…Many papers deal with the use of membrane adsorption for purification and isolation of proteins [26][27][28] and antibodies or to remove DNA, host cell proteins, endotoxins und viruses respectively [2,[7][8][9][10][11][12][13][14]. Membrane adsorbers employ the same strong anion and cation exchange ligands, quarternary ammonium (Q) respectively sulfonic acid (S), as standard ion exchange chromatography columns.…”