Details
| Original language | English |
|---|---|
| Pages (from-to) | 29-38 |
| Number of pages | 10 |
| Journal | Engineering in life sciences |
| Volume | 12 |
| Issue number | 1 |
| Publication status | Published - 23 Aug 2011 |
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 (42mg/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 200mg soluble FGF-2 was yielded from 1.9L 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.
Keywords
- Basic fibroblast growth factor, Escherichia coli BL21, Membrane adsorber technology, Pluripotent stem cell culture
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Environmental Science(all)
- Environmental Engineering
- Chemical Engineering(all)
- Bioengineering
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In: Engineering in life sciences, Vol. 12, No. 1, 23.08.2011, p. 29-38.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Cytokine production using membraneadsorbers: Human basic fibroblast growthfactor produced byEscherichia coli
AU - Chen, Ran
AU - John, Jinu
AU - Lavrentieva, Antonina
AU - Müller, Susann
AU - Tomala, Magda
AU - Zhao, Yangxi
AU - Zweigerdt, Robert
AU - Beutel, Sascha
AU - Hitzmann, Bernd
AU - Kasper, Cornelia
AU - Martin, Ulrich
AU - Rinas, Ursula
AU - Stahl, Frank
AU - Scheper, Thomas
PY - 2011/8/23
Y1 - 2011/8/23
N2 - 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 (42mg/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 200mg soluble FGF-2 was yielded from 1.9L 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.
AB - 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 (42mg/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 200mg soluble FGF-2 was yielded from 1.9L 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.
KW - Basic fibroblast growth factor
KW - Escherichia coli BL21
KW - Membrane adsorber technology
KW - Pluripotent stem cell culture
UR - http://www.scopus.com/inward/record.url?scp=84863011321&partnerID=8YFLogxK
U2 - 10.1002/elsc.201100045
DO - 10.1002/elsc.201100045
M3 - Article
AN - SCOPUS:84863011321
VL - 12
SP - 29
EP - 38
JO - Engineering in life sciences
JF - Engineering in life sciences
SN - 1618-0240
IS - 1
ER -