Details
| Original language | English |
|---|---|
| Pages (from-to) | 71-83 |
| Number of pages | 13 |
| Journal | Journal of biotechnology |
| Volume | 68 |
| Issue number | 1 |
| Publication status | Published - 5 Feb 1999 |
| Externally published | Yes |
Abstract
The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction elf the biomass yield coefficient with respect to glucose.
Keywords
- High-cell density cultivation, Human insulin, Recombinant Escherichia coli, Temperature-inducible expression
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Journal of biotechnology, Vol. 68, No. 1, 05.02.1999, p. 71-83.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli
AU - Schmidt, Michael
AU - Babu, Kunnel Raman
AU - Khanna, Navin
AU - Marten, Sabine
AU - Rinas, Ursula
PY - 1999/2/5
Y1 - 1999/2/5
N2 - The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction elf the biomass yield coefficient with respect to glucose.
AB - The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction elf the biomass yield coefficient with respect to glucose.
KW - High-cell density cultivation
KW - Human insulin
KW - Recombinant Escherichia coli
KW - Temperature-inducible expression
UR - http://www.scopus.com/inward/record.url?scp=0032970060&partnerID=8YFLogxK
U2 - 10.1016/S0168-1656(98)00189-8
DO - 10.1016/S0168-1656(98)00189-8
M3 - Article
C2 - 10036770
AN - SCOPUS:0032970060
VL - 68
SP - 71
EP - 83
JO - Journal of biotechnology
JF - Journal of biotechnology
SN - 0168-1656
IS - 1
ER -