Stabilization of single-chain Fv antibody production using recombinant Escherichia coli by DO-stat fed-batch culture employing yeast extract-enriched feeding medium
The stabilized production of anti-CRP single-chain Fv (scFv) antibody using recombinant E. coli by prolonged dissolved oxygen (DO)-stat fed-batch culture was demonstrated, focusing on the supply of nitrogen sources during fed-batch operation. The serious degradation of anti-CRP scFv by intracellular...
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Veröffentlicht in: | Biochemical engineering journal 2021-12, Vol.176, p.108184, Article 108184 |
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Sprache: | eng |
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Zusammenfassung: | The stabilized production of anti-CRP single-chain Fv (scFv) antibody using recombinant E. coli by prolonged dissolved oxygen (DO)-stat fed-batch culture was demonstrated, focusing on the supply of nitrogen sources during fed-batch operation. The serious degradation of anti-CRP scFv by intracellular proteases was observed when the fermentation time was prolonged from 50 h to 100 h, which was presumably induced by the insufficient supply of free amino acids. To evaluate the effects of amino acid supply on the proteolysis of anti-CRP scFv, fed-batch experiments using yeast extract (YE)-enriched feeding medium, which had various blend ratios of glucose and YE (G/YE), were performed. By increasing the YE content in the feeding medium, the expression of intracellular proteases was significantly suppressed; consequently, the proteolysis of the scFv production was successfully prevented in 100 h fed-batch culture. The desired G/YE ratio to avoid proteolysis was 1.0 g-glucose/g-YE. The stabilization of scFv production accordingly resulted in the enhanced scFv production from 3.0 g/L in 50 h fed-batch culture to 4.0–5.2 g/L in 100 h fed-batch culture with more than 97% solubility which corresponded to 94.9% of increase in total scFv production by one fed-batch operation.
•Serious proteolysis of single-chain Fv (scFv) was observed during fed-batch culture.•Use of yeast extract-enriched feeding medium prevented the proteolysis of scFv.•Stabilized production of 4.0–5.2 g/L of scFv was achieved by 100 h fed-batch cultivation. |
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ISSN: | 1369-703X 1873-295X |
DOI: | 10.1016/j.bej.2021.108184 |