Details
| Original language | English |
|---|---|
| Pages (from-to) | 235-241 |
| Number of pages | 7 |
| Journal | Enzyme and microbial technology |
| Volume | 34 |
| Issue number | 3-4 |
| Publication status | Published - Mar 2004 |
| Externally published | Yes |
Abstract
Many recombinant proteins partially aggregate into inclusion bodies during production in Escherichia coli in batch culture on defined medium. Production on complex medium, however, effectively prevented inclusion body formation of a β-galactosidase-HIVgp41 fusion protein for detection of anti-HIV antibodies which is produced at 42°C under control of a temperature- inducible expression system. Cells pre-conditioned by cultivation on complex medium before induction showed faster growth, higher product concentration and reduced inclusion body formation even when producing on defined medium. In contrast, for human basic fibroblast growth factor (hFGF-2) produced under control of the phage T7-promoter, medium composition could not reduce inclusion body formation even at 30°C. Here, slow production in high-cell density fed-batch mode using a defined medium with limited glucose feeding enabled the accumulation of 50mg product per gram cell dry mass exclusively in the soluble cell fraction, resulting in a volumetric concentration of more than 4g per litre hFGF-2. With the β-galactosidase fusion protein produced in fed-batch, over 100mg of product per gram cell dry mass accumulated in the soluble cell fraction. With a cell density of 100g cell dry mass per litre, this resulted in a volumetric concentration of 10g per litre of soluble β-galactosidase-HIVgp41 fusion protein. Thus, two approaches to balance heterologous protein production and host physiology are presented, which fit the needs of lab bench or pilot plant, respectively.
Keywords
- Cultivation, Escherichia coli, hFGF-2, High-cell density, Inclusion bodies, Recombinant protein production, β-galactosidase fusion protein
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
Sustainable Development Goals
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In: Enzyme and microbial technology, Vol. 34, No. 3-4, 03.2004, p. 235-241.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale
AU - Hoffmann, Frank
AU - Van Den Heuvel, Joop
AU - Zidek, Nadine
AU - Rinas, Ursula
PY - 2004/3
Y1 - 2004/3
N2 - Many recombinant proteins partially aggregate into inclusion bodies during production in Escherichia coli in batch culture on defined medium. Production on complex medium, however, effectively prevented inclusion body formation of a β-galactosidase-HIVgp41 fusion protein for detection of anti-HIV antibodies which is produced at 42°C under control of a temperature- inducible expression system. Cells pre-conditioned by cultivation on complex medium before induction showed faster growth, higher product concentration and reduced inclusion body formation even when producing on defined medium. In contrast, for human basic fibroblast growth factor (hFGF-2) produced under control of the phage T7-promoter, medium composition could not reduce inclusion body formation even at 30°C. Here, slow production in high-cell density fed-batch mode using a defined medium with limited glucose feeding enabled the accumulation of 50mg product per gram cell dry mass exclusively in the soluble cell fraction, resulting in a volumetric concentration of more than 4g per litre hFGF-2. With the β-galactosidase fusion protein produced in fed-batch, over 100mg of product per gram cell dry mass accumulated in the soluble cell fraction. With a cell density of 100g cell dry mass per litre, this resulted in a volumetric concentration of 10g per litre of soluble β-galactosidase-HIVgp41 fusion protein. Thus, two approaches to balance heterologous protein production and host physiology are presented, which fit the needs of lab bench or pilot plant, respectively.
AB - Many recombinant proteins partially aggregate into inclusion bodies during production in Escherichia coli in batch culture on defined medium. Production on complex medium, however, effectively prevented inclusion body formation of a β-galactosidase-HIVgp41 fusion protein for detection of anti-HIV antibodies which is produced at 42°C under control of a temperature- inducible expression system. Cells pre-conditioned by cultivation on complex medium before induction showed faster growth, higher product concentration and reduced inclusion body formation even when producing on defined medium. In contrast, for human basic fibroblast growth factor (hFGF-2) produced under control of the phage T7-promoter, medium composition could not reduce inclusion body formation even at 30°C. Here, slow production in high-cell density fed-batch mode using a defined medium with limited glucose feeding enabled the accumulation of 50mg product per gram cell dry mass exclusively in the soluble cell fraction, resulting in a volumetric concentration of more than 4g per litre hFGF-2. With the β-galactosidase fusion protein produced in fed-batch, over 100mg of product per gram cell dry mass accumulated in the soluble cell fraction. With a cell density of 100g cell dry mass per litre, this resulted in a volumetric concentration of 10g per litre of soluble β-galactosidase-HIVgp41 fusion protein. Thus, two approaches to balance heterologous protein production and host physiology are presented, which fit the needs of lab bench or pilot plant, respectively.
KW - Cultivation
KW - Escherichia coli
KW - hFGF-2
KW - High-cell density
KW - Inclusion bodies
KW - Recombinant protein production
KW - β-galactosidase fusion protein
UR - http://www.scopus.com/inward/record.url?scp=0442292087&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2003.10.011
DO - 10.1016/j.enzmictec.2003.10.011
M3 - Article
AN - SCOPUS:0442292087
VL - 34
SP - 235
EP - 241
JO - Enzyme and microbial technology
JF - Enzyme and microbial technology
SN - 0141-0229
IS - 3-4
ER -