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Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression

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Abstract

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, 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 scFv with enhanced green fluorescent protein (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 as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

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Acknowledgements

We thank Sandra Baumann and Kai Scharnweber, Institute of Biochemical Engineering, University of Stuttgart, for their technical assistance in cell-free protein biosynthesis; Volker Noedinger, Institute of Technical Biochemistry, University of Stuttgart, for N-terminal protein sequencing; Dr. Annett Burzlaff, Institute of Cell Biology and Immunology, University of Stuttgart, for technical support with microscopy; and Dr. Karl Kramer and Dr. Berthold Hock, Technical University of Muenchen at Weihenstephan, for the plasmid pCANTAB 5E. Financial support by the "Forschungsschwerpunkt Biosystemtechnik des Landes Baden-Wuerttemberg" is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 , Stuttgart, Germany

    P. Oelschlaeger, S. Lange, J. Schmitt & R. D. Schmid

  2. Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 , Stuttgart, Germany

    M. Siemann & M. Reuss

Authors
  1. P. Oelschlaeger
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  2. S. Lange
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  3. J. Schmitt
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  4. M. Siemann
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  5. M. Reuss
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  6. R. D. Schmid
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Corresponding author

Correspondence to R. D. Schmid.

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For correspondence with regard to cell-free protein synthesis: Dr. Martin Siemann (e-mail: siemann@ibvt.uni-stuttgart.de, Tel.: +49-711-6855161, Fax: +49-711-6855164)

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Oelschlaeger, P., Lange, S., Schmitt, J. et al. Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coli by comparing in vivo and in vitro expression. Appl Microbiol Biotechnol 61, 123–132 (2003). https://doi.org/10.1007/s00253-002-1190-6

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  • DOI: https://doi.org/10.1007/s00253-002-1190-6

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