Expression of a recombinant human anti-MUC1 scFv fragment in protease-deficient Escherichia coli mutants

Kandilogiannaki,M.; Koutsoudakis,G.; Zafiropoulos,A.; Krambovitis,E.

doi:10.3892/ijmm.7.6.659

June 2001 Volume 7 Issue 6

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June 2001 Volume 7 Issue 6

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Article

Expression of a recombinant human anti-MUC1 scFv fragment in protease-deficient Escherichia coli mutants

Authors:
- M. Kandilogiannaki
- G. Koutsoudakis
- A. Zafiropoulos
- E. Krambovitis
View Affiliations / Copyright

Affiliations: Department of Applied Biochemistry and Immunology, Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Crete, Greece
Pages: 659-664
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Published online on: June 1, 2001

https://doi.org/10.3892/ijmm.7.6.659
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Abstract

The proteolytic activity of Escherichia coli periplasmic proteases can affect the expression efficiency of many heterologous proteins such as antibody fragments that are transported to the host periplasm for folding. We investigated whether four E. coli strains that were deficient in the periplasmic proteases tsp, protease III, degP and ompT, in different combinations, affect the expression levels of an anti-MUC1 scFv fragment. The ompT protease appeared to be involved in partial degradation of the scFv since degradation products were observed in all ompT unmutated strains in Western blotting, whereas such products were absent in the ompT mutated strains. The HM120 strain that contained most mutations, expressed the scFv protein efficiently but the level of functional antibody activity was low. This was probably due to an accumulation of incorrectly folded antibody molecules in the periplasm as it was characterised by low enzyme immunoassay reactions in contrast to the intense staining of the tag in Western blots. Improved understanding of the periplasmic protease involvement in the process of the antibody expression in bacteria may allow us to design host E. coli strains that are more efficient in producing functional antibodies.

Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Expression of a recombinant human anti-MUC1 scFv fragment in protease-deficient Escherichia coli mutants

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