Please use this identifier to cite or link to this item: http://dx.doi.org/10.18419/opus-1686
Schmid, Rolf D.
|Title:||Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression|
|metadata.ubs.publikation.source:||Applied microbiology & biotechnology 61 (2003), S. 123-132. URL http://dx.doi.org./10.1007/s00253-002-1190-6|
|Abstract:||In order to produce the atrazine-specific scFv K411B, it was expressed in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the scFv was N-terminally fused to the pelB leader, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly: The expression level of the scFv with leader was 2.3 times higher than that of the protein without leader. To further investigate this, the respective translation profiles were generated by coupled in vitro transcription/translation assays and gave according results. Periplasmic expression resulted in only 10% correctly folded scFv. The same percentage was obtained when the scFv was expressed in vitro, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of the scFv with EGFP fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.|
|Appears in Collections:||04 Fakultät Energie-, Verfahrens- und Biotechnik|
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