Details
| Original language | English |
|---|---|
| Pages (from-to) | 947-953 |
| Number of pages | 7 |
| Journal | Enzyme and microbial technology |
| Volume | 17 |
| Issue number | 10 |
| Publication status | Published - 28 Dec 1999 |
| Externally published | Yes |
Abstract
Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escherichia coli TGl as host organism. The bFGF structural gene was cloned into two vectors differing only with respect to the promoter, which was either the bacteriophage λ PRPL- or the E. coli lac-promoter. The resulting expression systems were studied in high-cell density cultures. Cells were grown in a fed-batch procedure with a predetermined exponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30°C. Product formation was induced by either a temperature shift from 30 to 42°C or by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). Under comparable culture conditions-induction of bFGF expression at 41 to 45 g l-1 dry cell weight and the addition of feed medium with identical rates-bFGF accumulated to 4.9 and 1.1 g l-1 using the temperature- and IPTG-inducible expression systems, respectively. The final biomass concentrations obtained using temperature- and IPTG-inducible expression systems were 61 and 135 g l-1 dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g-1 dry cell weight, respectively. When a temperature shift was used for product induction, 30% of bFGF was recovered as inclusion bodies in the insoluble cell fraction. IPTG-dependent induction yielded exclusively soluble bFGF.
Keywords
- Basic fibroblast growth factor, comparison of different expression systems, high-cell-density cultivation, recombinant Escherichia coli
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
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In: Enzyme and microbial technology, Vol. 17, No. 10, 28.12.1999, p. 947-953.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Comparison of temperature- and isopropyl-β-d-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli
AU - Seeger, Anke
AU - Schneppe, Bernard
AU - McCarthy, John E.G.
AU - Deckwer, Wolf Dieter
AU - Rinas, Ursula
PY - 1999/12/28
Y1 - 1999/12/28
N2 - Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escherichia coli TGl as host organism. The bFGF structural gene was cloned into two vectors differing only with respect to the promoter, which was either the bacteriophage λ PRPL- or the E. coli lac-promoter. The resulting expression systems were studied in high-cell density cultures. Cells were grown in a fed-batch procedure with a predetermined exponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30°C. Product formation was induced by either a temperature shift from 30 to 42°C or by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). Under comparable culture conditions-induction of bFGF expression at 41 to 45 g l-1 dry cell weight and the addition of feed medium with identical rates-bFGF accumulated to 4.9 and 1.1 g l-1 using the temperature- and IPTG-inducible expression systems, respectively. The final biomass concentrations obtained using temperature- and IPTG-inducible expression systems were 61 and 135 g l-1 dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g-1 dry cell weight, respectively. When a temperature shift was used for product induction, 30% of bFGF was recovered as inclusion bodies in the insoluble cell fraction. IPTG-dependent induction yielded exclusively soluble bFGF.
AB - Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escherichia coli TGl as host organism. The bFGF structural gene was cloned into two vectors differing only with respect to the promoter, which was either the bacteriophage λ PRPL- or the E. coli lac-promoter. The resulting expression systems were studied in high-cell density cultures. Cells were grown in a fed-batch procedure with a predetermined exponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30°C. Product formation was induced by either a temperature shift from 30 to 42°C or by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). Under comparable culture conditions-induction of bFGF expression at 41 to 45 g l-1 dry cell weight and the addition of feed medium with identical rates-bFGF accumulated to 4.9 and 1.1 g l-1 using the temperature- and IPTG-inducible expression systems, respectively. The final biomass concentrations obtained using temperature- and IPTG-inducible expression systems were 61 and 135 g l-1 dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g-1 dry cell weight, respectively. When a temperature shift was used for product induction, 30% of bFGF was recovered as inclusion bodies in the insoluble cell fraction. IPTG-dependent induction yielded exclusively soluble bFGF.
KW - Basic fibroblast growth factor
KW - comparison of different expression systems
KW - high-cell-density cultivation
KW - recombinant Escherichia coli
UR - http://www.scopus.com/inward/record.url?scp=0028970554&partnerID=8YFLogxK
U2 - 10.1016/0141-0229(94)00123-9
DO - 10.1016/0141-0229(94)00123-9
M3 - Article
AN - SCOPUS:0028970554
VL - 17
SP - 947
EP - 953
JO - Enzyme and microbial technology
JF - Enzyme and microbial technology
SN - 0141-0229
IS - 10
ER -