High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum
We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum . For efficient secretory production of the antibody fragment, the following components were examined: (1) s...
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Veröffentlicht in: | Applied microbiology and biotechnology 2014-01, Vol.98 (1), p.273-284 |
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Sprache: | eng |
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Zusammenfassung: | We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in
Corynebacterium glutamicum
. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5′ untranslated region (5′ UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD
600
of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in
C. glutamicum
. |
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ISSN: | 0175-7598 1432-0614 |
DOI: | 10.1007/s00253-013-5315-x |